JP2011041573A - Composition and method for enhancing synthesis of nucleic acid molecule - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide compositions and methods for enhancing synthesis of nucleic acid molecules, particularly GC-rich nucleic acid molecules. <P>SOLUTION: The compositions comprise one or more nitrogen-containing organic compounds selected from the group consisting of specific constitutional formulae (or salts or derivatives thereof), preferably 4-methylmorpholine N-oxide or betaine (carboxymethyltrimethylammonium), and further comprises one or more compounds selected from the group consisting of proline and an N-alkylimidazole compound, and more preferably proline, 1-methylimidazole or 4-methylimidazole. The invention further relates to methods for high-fidelity synthesis of nucleic acid molecules, including via amplification, reverse transcription, and sequencing. The invention also relates to nucleic acid molecules synthesized by these methods or derivatives thereof, and to vectors and host cells comprising such nucleic acid molecules or derivatives. The invention also relates to kits for synthesizing, amplifying, reverse transcribing, or sequencing nucleic acid molecules comprising one or more of the compositions. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

(Background of the Invention)
(Field of Invention)
The present invention is in the fields of molecular biology and cell biology. The present invention relates generally to compounds, compositions and methods useful for enhancing the synthesis of nucleic acid molecules, particularly GC-rich nucleic acid templates. In particular, the present invention provides a composition comprising one or more compounds having a formula selected from the group consisting of Formula I and Formula II. Preferably, 4-methylmorpholine N-oxide, betaine (carboxymethyltrimethylammonium), any amino acid (or derivative thereof), and / or N-alkylimidazole (eg 1-methylimidazole or 4-methylimidazole) is present. Used according to the invention. In preferred aspects, two or more, three or more, four or more, etc. compounds of the invention are combined to facilitate nucleic acid synthesis.

  The invention also includes one or more compounds of the invention, and (i) one or more nucleic acid molecules (including nucleic acid templates), (ii) one or more nucleotides, (iii) one or more polymerases or It relates to a composition comprising reverse transcriptase, and (iv) one or more additional components selected from the group consisting of one or more buffer salts.

  These compounds and compositions of the invention can be used in methods for enhanced, high fidelity synthesis of nucleic acid molecules, including methods by amplification (particularly PCR), reverse transcription, and sequencing. The invention also relates to nucleic acid molecules, fragments or derivatives thereof produced by these methods, and vectors and host cells containing such nucleic acid molecules, fragments or derivatives. The invention also relates to the use of such nucleic acid molecules to produce the desired polypeptide. The invention also relates to a kit comprising a composition or compound of the invention.

(Related fields)
(Genomic DNA)
When examining the structure and physiology of an organism, tissue or cell, it is often desirable to determine their genetic content. The genetic backbone (ie, genome) of an organism is encoded in a double-stranded sequence of nucleotide bases in deoxyribonucleic acid (DNA) contained in the somatic and germ cells of the organism. A particular segment of DNA, ie the genetic content of a gene, is expressed only by the production of the protein that this gene ultimately encodes. To produce a protein, a complementary copy (“sense” strand) of one strand of a DNA double helix is generated by the polymerase enzyme, resulting in the specific sequence of messenger ribonucleic acid (mRNA). Arise. This mRNA is then translated by the cellular protein synthesis machinery, resulting in the production of the specific protein encoded by this gene. There are additional sequences in the genome (ie, “non-coding” regions) that do not encode proteins that can play a structural, regulatory or unknown function role. Thus, the genome of an organism or cell is a complete collection of protein-coding genes along with intervening non-coding DNA sequences. Importantly, each somatic cell in a multicellular organism, except in the case of focal infection or cancer, contains a complete complement of the organism's genomic DNA, where one or more heterologous DNA sequences are identified. May be inserted into the genomic DNA of other cells and may not be inserted into other non-infected cells of the organism. However, as described below, the expression of the genes making up the genomic DNA can vary between individual cells.

(CDNA and cDNA library)
There are a myriad of mRNA species within a given cell, tissue, or organism, each encoding a separate and specific protein. This fact provides a powerful tool for researchers interested in studying gene expression in tissues or cells. mRNA molecules can be isolated and further manipulated by a variety of molecular biology techniques, thereby allowing an assessment of the fully functional gene content of a cell, tissue, or organism.

  One common approach to gene expression studies is the production of complementary DNA (cDNA) clones. In this technique, mRNA molecules from an organism are isolated from extracts of the organism's cells or tissues. This isolation often utilizes a solid phase chromatography matrix (eg, cellulose or hydroxyapatite) to which an oligomer of deoxythymidine (dT) is complexed. Since the 3 ′ end on all eukaryotic mRNA molecules contains a stretch of deoxyadenosine (dA) bases and dA binds to dT, the mRNA molecule is another molecule and substance in tissue or cell extracts. Can be rapidly purified. From these purified mRNA molecules, cDNA copies can be made using the enzyme reverse transcriptase (reverse transcriptase). This produces a single stranded cDNA molecule. The single stranded cDNA is then converted into a complete double stranded DNA copy of the original mRNA (and thus the original double stranded DNA sequence encoding this mRNA, which is contained in the organism's genome). It can be converted by the action of a polymerase. The protein specific double stranded cDNA can then be inserted into a plasmid, which is then introduced into the host bacterial cell. The bacterial cells are then grown in culture medium, resulting in a population of bacterial cells containing (or often expressing) the gene of interest.

  This entire process (from isolation of mRNA to insertion of cDNA into a plasmid to propagation of a bacterial population containing the isolated gene) is called “cDNA cloning”. When cDNA is prepared from many different mRNAs, the resulting set of cDNAs is referred to as a “cDNA library” and is different functional (ie, expressed) present in the source cell, tissue, or organism. Represents a gene. Genotyping of these cDNA libraries can produce a lot of information about the structure and function of the organism from which they are derived.

(DNA amplification)
Researchers have relied on many amplification techniques to increase, or “amplify,” the copy number of a particular sequence of DNA in a sample. Commonly used amplification techniques include the polymerase chain reaction (“PCR”) method described by Mullis and co-workers (US Pat. Nos. 4,683,195; 4,683,202; and No. 4,800,159). This method uses “primer” sequences that are complementary to opposing regions on the DNA sequence to be amplified. These primers are added to the DNA target sample with a molar excess of nucleotide bases and DNA polymerase (eg, Taq polymerase), and the primers are base-specific binding interactions (ie, adenine is thymine) to their targets. In addition, cytosine binds via guanine). Repeat the reaction mixture through cycles of increasing and decreasing temperature (allowing dissociation of the two DNA strands on the target sequence, synthesis of complementary copies of each strand by polymerase, and reannealing of the new complementary strand) The number of copies of a particular sequence of DNA can be rapidly increased.

  Other techniques for amplification of target nucleic acid sequences have also been developed. For example, Walker et al. (US Pat. No. 5,455,166; EP 0684315) describe a method called Strand Displacement Amplification (SDA), which is operated at a single temperature and is a single target DNA sequence. It differs from PCR in that it uses an enzyme polymerase / endonuclease combination that produces a strand fragment, which in turn serves as a template to produce a complementary DNA (cDNA) strand. An alternative amplification procedure called Nucleic Acid Sequence-Based Amplification (NASBA) is disclosed in Davey et al. (US Pat. No. 5,409,818; EP0329822). Like SDA, NASBA uses an isothermal reaction but is based on the use of RNA primers for amplification rather than DNA primers as in PCR or SDA. Another known amplification procedure includes the promoter ligated activated transcriptase (LAT) described by Berninger et al. (US Pat. No. 5,194,370).

(DNA fingerprinting based on PCR)
Despite the availability of various amplification techniques, most DNA fingerprinting methods rely on PCR for amplification and utilize well-characterized protocols and automation available for this technique. Examples of these PCR-based fingerprinting techniques include Random Amplified Polymorphic DNA (RAPD) analysis (Williams, JGK, et al., Nucl. Acids Res. 18 (22): 6531-6535 (1990)), Arbitrary Primed PCR (AP-PCR; Welsh, J., and McClelland, M., Nucl. Acids Res. 18 (24): 7213-7218 (1990)) , DNA Amplification Fingerprinting (DAF; Caetano-Anoles et al., Bio / Technology 9: 553). -557 (1991)), and microsatellite PCR or direct amplification of minisatellite-region DNA (DAMD; Heath, DD et al., Nucl. Acids Res. 21 (24): 5782-5785 (1993)). All of these methods are based on amplification of random DNA fragments by PCR using arbitrarily selected primers.

(DNA sequencing)
In general, two techniques have traditionally been used to sequence nucleic acids. The first called “Maxam and Gilbert Sequencing” after its co-developer (Maxam, AM and Gilbert, W., Proc. Natl. Acad. Sci. USA 74: 560-564, 1977) In the method, DNA is radiolabeled, divided into four samples, and treated with chemicals that selectively destroy specific nucleotide bases in the DNA and cleave molecules at the site of damage. By separating the resulting fragments into distinct bands by gel electrophoresis and exposing the gel to X-ray film, the original DNA molecule sequence can be read from the film. This technique involves the use of certain complex DNA molecules (primate virus SV40 (Fiers, W. et al., Nature 273: 113-120, 1978; Reddy, V.B., et al., Science 200: 494-502, 1978), And the sequence of bacterial plasmid pBR322 (including Sutcliffe, G., Cold Spring Harbor Symp. Quant. Biol. 43: 444-448, 1975).

  Alternative for sequencing named after its developer (Sanger, F., and Coulson, AR, J. Mol. Biol. 94: 444, 448, 1975) This technique is more commonly used. This method uses the DNA synthesis activity of DNA polymerase, which is a reaction terminated dideoxynucleoside triphosphate (Sanger, F., et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467, 1977) and short. When combined with a mixture of primers, any of which can be detectably labeled, yields a series of newly synthesized DNA fragments that are specifically terminated with one of the four dideoxy bases. These fragments are then separated by gel electrophoresis and the sequence is determined as described for the Maxam and Gilbert sequencing method described above. By performing four separate reactions (once for each ddNTP), even a fairly complex sequence of DNA molecules can be rapidly determined (Sanger, F., et al., Nature 265: 678-695, 1977; Barnes). , W., Meth. Enzymol. 152: 538-556, 1987). Sanger's sequencing method is generally described in E. E. coli or T7 DNA polymerase (US Pat. No. 4,795,699), but recent modifications of this technique using T7 polymerase mutants are that sequencing determines that all four chain-terminated ddNTPs have different concentrations. In a single sequencing reaction (US Pat. Nos. 4,962,020 and 5,173,411). Further modifications to this technique to reduce or eliminate the accumulation of pyrophosphate that would harm the reaction in the reaction mixture have also been described (US Pat. No. 5,498,523). Other modifications for sequencing nucleic acid molecules have also been described (Murray, Nucl. Acids. Res. 17: 8889, 1989; and Craxton, Methods: A Comparison to Methods in Enzymology, 3: 20-25, 1991).

(Limited)
As noted above, reliable and high fidelity replication of template nucleic acid molecules is an essential step in the synthesis of nucleic acid molecules in amplification, reverse transcription, and sequencing protocols. However, the use of standard compositions and protocols to accomplish this synthesis is often inefficient in the following respects: they are specific secondary structures in the nucleic acid template (Gerald, GF, et al. , FOCUS 11 (4): 60 (1989); Myers, TW, and Gelfund, DH, Biochemistry 30: 7661 (1991)) barriers and sequences (Messer, LI, et al., Virol). 146: 146 (1985)); Abbotts, J. et al. , Et al. Biol. Chem. 268: 10312-10323 (1993)) There is a tendency to terminate nucleic acid synthesis unfinished by a barrier. This is especially true for template sequences with high guanidine / cytosine content (ie, “GC-rich” templates) and fairly large sized template sequences (ie, templates longer than about 3-5 kb long). These secondary structure and sequence barriers in template nucleic acid molecules frequently occur with homopolymer stretches (Messer. LI et al., Virol. 146: 146 (1985)); Huber, H. et al. E. , Et al. Biol. Chem. 264: 4669-4678 (1989); Myers, T .; W. , And Gelfund, D .; H. , Biochemistry 30: 7661 (1991)) and sequence barriers are more frequent than secondary structure barriers (Abbotts, J., et al., J. Biol. Chem. 268: 10312-10323 (1993)). If these barriers can be overcome, the yield of total and full-length nucleic acid products in the synthesis reaction can be increased.

Adjustment of the ionic strength or osmotic pressure of the reaction mixture, in particular the concentration of Na ⁺ and K ⁺ ions, is as in vivo (LeRudulier, D., et al., Science 224: 1064 (1984); Buche, A., et al. J. Biomolec.Struct.Dyn.8 (3): 601 (1990); Marquet, R., and Housesier, C., J. Biomolec.Struct.Dyn.9 (1): 159 (1991); A., et al., J. Biomolec. Struct.Dyn.11 (1): 95 (1993); Woodford, K., et al., Nucl. Acids Res.23 (3): 539 (1995); Flock, S. et al. Biophys.J.70: 1456 (1996); Et al, Biophys.J.71: 1519 (1996); EP0821059A2), that in vitro may influence the secondary structure and condensation of the nucleic acid, several reports have shown. In some of these studies, in vitro nucleic acid conformation and stability has been demonstrated by polysaccharides such as trehalose (Carninci, P., et al., Proc. Natl. Acad. Sci. USA 95: 520-524 (1998)). Certain cosolvents such as glycerol and dimethyl sulfoxide (Varadaraj, K., and Skinner, DM, Gene 140: 1 (1994)); glycine and its derivatives (Buche, A., et al., FEBS Lett. 247 (2): 367 (1989); Flock, S., et al., J. Biomolec. Struct. Dyn. 13 (1): 87 (1995), Housesier, C., et al., Comp. Biochem. 3): 313 1997)); low molecular weight amines such as β-alanine, asparagine, and cystamine (Kondakova, NV, et al., Mol. Biol. (Moscow) 9 (5): 742 (1975); Aslanian, VM. , Et al., Biofizika 29 (4): 564 (1984)); and other nitrogen-containing compounds and amino acids such as proline, betaine, and ectoine (Rees, WA, et al., Biochemistry 32: 137- 144 (1993); WO95 / 20682; DE4411588C1; DE4411594C1; Mytelka, DS, et al., Nucl. Acids Res.24 (14): 2774 (1996); Baskaran, N., et al., Genome Res.6. 633 (1996); Weissenstainer, T., and Ranchbury, JS, BioTechniques 21 (6): 1102 (1996); Rajendrakumar, CSV, et al., FEBS Letts. 410: 201-205 (1997). Henke, W., et al., Nucl. Acids Res. 25 (19): 3957 (1997); Hengen, PN TIBS 22: 225 (1997))) It was found to be improved in a buffer solution containing any of the following. Betaine and ectoine are natural osmoprotectants in various bacterial and animal cells (Chambers, ST, et al., J. Bacteriol. 169 (10): 4845 (1987); Randall, K., Biochim.Biophys.Acta 1291 (3): 189 (1996); Randall, K., et al., Biochem.Cell Biol.74 (2): 283 (1996); Malin, G., and Lapidot, A.,. J. Bacteriol.178 (2): 385 (1996); Gouesbet, G., et al., J. Bacteriol.178 (2): 447 (1996); Canovas, D., et al., J.Bacteriol.178 (24) : 7221 (1996); Canov . S, D, et al., J.Biol.Chem.272 (41): 25794-25801 (1997).

  However, there is a need in the art for compounds, compositions, and methods that are useful for enhancing the synthesis of nucleic acid molecules, particularly GC-rich and / or relatively large nucleic acid molecules.

(Simple Summary of Invention)
The present invention relates generally to compounds, compositions and methods useful for enhancing the synthesis of nucleic acid molecules, particularly nucleic acid molecules derived from GC-rich nucleic acid templates. In one aspect, the invention relates to compounds and compositions for use in the synthesis of nucleic acid molecules, particularly for template-mediated synthesis, such as amplification, reverse transcription, and sequencing reactions. The compounds and compositions of the present invention include one or more compounds having a chemical formula selected from the group consisting of Formula I and Formula II, and salts or derivatives thereof. In one preferred aspect, the compounds used in the present invention include any amino acid, any saccharide (monosaccharide or polysaccharide), any polyalcohol, or a salt or derivative thereof. The compound or composition of the present invention includes a compound having the chemical formula shown in Formula I or Formula II, or a salt or derivative thereof, wherein the aryl group includes a phenyl group, a naphthyl group, a phenanthryl group, an anthracyl group. Selected from the group consisting of a group, an indenyl group, an azulenyl group, a biphenyl group, a biphenylenyl group, and a fluorenyl group; wherein the halo group is selected from the group consisting of fluorine, chlorine, bromine, and iodine; The alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, and decyl and can be a branched chain alkyl group; The group includes ethenyl group, propenyl group, butenyl group, pentenyl group, hexenyl group, Selected from the group consisting of a ptenyl group, an octenyl group, a nonenyl group, and a decynyl group, and may be a branched alkenyl group; where the alkenyl group is an ethynyl group, a propynyl group, a butynyl group, a pentynyl group, a hexynyl group Selected from the group consisting of a group, a heptynyl group, an octynyl group, a noninyl group, and a decynyl group, and may be a branched alkynyl group; and wherein the lower alkoxy (ether) group is one of the above alkyl groups Is replaced by oxygen. The invention also relates to salts and derivatives of such compounds. In a particularly preferred aspect of the invention, the compound is 4-methylmorpholine N-oxide, betaine, carnitine, ectoine, proline, glycine, pipecolic acid, trimethylamine N oxide, N-alkylimidazole compounds (eg 1-methylimidazole or 4 -Methylimidazole), poly (2-ethyl-2-oxazoline) having an average molecular weight of about 50,000 to about 500,000 daltons, poly (diallyldimethylammonium chloride) having an average molecular weight of about 100,000 to about 200,000 daltons, Or selected from the group consisting of salts or derivatives thereof. The invention also provides a compound of the invention, and (i) one or more enzymes having nucleic acid polymerase activity that can be a thermostable enzyme, (ii) one or more nucleotides, (iii) one or more buffered salts, And (iv) a composition comprising one or more additional components selected from the group consisting of one or more nucleic acid molecules. Preferred such enzymes according to this aspect of the invention include DNA polymerases (eg, Taq, Tne, Tma, Pfu, VENT ^™ , DEEPVENT ^™ , and Tth DNA polymerases, and variants, variants, and derivatives thereof) , RNA polymerases (eg, SP6, T7, or T3 RNA polymerases, and variants, variants, and derivatives thereof) and reverse transcriptases (eg, M-MLV reverse transcriptase, RSV reverse transcriptase, AMV reverse transcriptase) Enzymes, RAV reverse transcriptase, MAV reverse transcriptase, and HIV reverse transcriptase, and variants, variants, and derivatives thereof). Preferably, such reverse transcriptase has reduced or substantially reduced RNase H space activity.

  The invention also relates to a method for synthesizing a nucleic acid molecule comprising: (a) a nucleic acid template (which can be a DNA molecule such as a cDNA molecule or an RNA molecule such as an mRNA molecule) and one or more ( Preferably 2 or more, 3 or more, 4 or more, 5 or more, etc.) compound or composition of the present invention to form a mixture; and (b) the mixture of the template Incubating under conditions sufficient to produce a first nucleic acid molecule that is complementary in whole or in part. Such methods of the invention optionally include one or more additional steps (eg, the first nucleic acid molecule described above is complementary to all or part of the first nucleic acid molecule). Incubating under conditions sufficient to produce a second nucleic acid molecule). The invention also relates to nucleic acid molecules produced by these methods, vectors containing these nucleic acid molecules (which can be expression vectors), and host cells containing these nucleic acid molecules or vectors. The present invention also relates to a method of producing a polypeptide, the method comprising culturing the above host cell under conditions convenient for production of the polypeptide by the host, and isolating the polypeptide. Process. The invention also relates to polypeptides produced by such methods.

The present invention also relates to a method for amplifying a nucleic acid molecule, the method comprising: (a) mixing a nucleic acid template with one or more compounds or compositions of the present invention to form a mixture; and (b) ) Incubating the mixture under conditions sufficient to amplify nucleic acid molecules complementary to all or part of the template. More particularly, the present invention relates to a method for amplifying a DNA molecule, the method comprising:
(A) providing first and second primers, wherein the first primer is complementary to a sequence at or near the 3 ′ end of the first strand of the DNA molecule; And the second primer is complementary to a sequence at or near the 3 ′ end of the second strand of the DNA molecule;
(B) the first strand in the first strand and the second primer in the presence of one or more compounds or compositions of the invention against the second strand. Hybridizing under conditions such that a third DNA molecule complementary to and a fourth DNA complementary to this second strand are synthesized;
(C) a step of denaturing the first strand and the third strand, and the second strand and the fourth strand; and (d) a step of repeating steps (a) to (c) one or more times,
Is included.

  Such conditions can include incubation in the presence of one or more polymerases, one or more nucleotides and / or one or more buffering salts. The invention also relates to nucleic acid molecules amplified by these methods.

  The invention also includes the following steps: (a) a nucleic acid molecule to be sequenced, one or more primers, one or more compounds or compositions of the invention, one or more nucleotides, and one or more Mixing with a terminator to form a mixture; (b) incubating the mixture under conditions sufficient to synthesize a population of molecules complementary to all or part of the molecule to be sequenced And (c) separating the population to determine the nucleotide sequence of all and part of the molecule to be sequenced, and a method for sequencing a nucleic acid molecule. More particularly, the invention relates to a method for sequencing a DNA molecule, the method comprising the following steps: (a) hybridizing a primer to the first DNA molecule; (b) the above steps Contacting the molecule of (a) with deoxyribonucleoside triphosphate, one or more compounds or compositions of the invention, and one or more terminator nucleotides; (c) mixing the mixture of step (b) above Incubating under conditions sufficient to synthesize with a random population of DNA molecules complementary to a DNA molecule, wherein the synthesized DNA molecule is longer than the first DNA molecule. The synthesized DNA molecule comprises a terminator nucleotide at its 3 ′ end; and (d) the synthesized DNA molecule is By separating the figure, the first at least step portion can be determined for the nucleotide sequence of the DNA molecule.

  Such terminator nucleotides include ddNTP, ddATP, ddGTP, ddITP, or ddCTP. Such conditions can include incubation in the presence of one or more DNA polymerases and / or buffering salts.

  The present invention also relates to a kit for use in the synthesis of nucleic acid molecules, the kit comprising one or more containers containing one or more compounds or compositions of the invention. These kits of the invention optionally contain one or more nucleotides, one or more polymerases, and / or reverse transcriptase, a suitable buffer, one or more primers, and one or more terminators. One or more additional components selected from the group consisting of (eg, one or more dideoxynucleotides) may be included.

  Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following drawings and description of the invention, and of the claims.

FIG. 1 shows ethidium bromide staining of samples of PCR reaction mixtures amplified in the presence of the indicated concentrations of proline, 1-methylimidazole, 4-methylimidazole, betaine, or in the absence of any of these cosolvents. It is a photograph of an agarose gel. M: DNA size marker. FIG. 2 is a photograph of an ethidium bromide stained agarose gel of a sample of a PCR reaction mixture amplified in the presence of the indicated concentrations of betaine or MMNO. M: DNA size marker. FIG. 3 is a photograph of an ethidium bromide stained agarose gel of samples of amplification of three different Pseudomonas aeruginosa amplicons (AprD, AprE and AprF) in the presence or absence of various compound combinations. Lane 1: 1 M betaine; Lane 2 1M TMANO; Lane 3-7: 2M (lane 3), 1M (lane 4), 0.5M (lane 6), 0.4M (lane 6), or 0.2M (lane 7) MMNO; lane 8: control without compound, M: DNA size marker. FIG. 4 is a photograph of an ethidium bromide stained agarose gel of a sample of PCR amplification of p53 exon 10 under different reaction buffer conditions in the presence or absence of the indicated concentrations of betaine, MMNO or proline. FIG. 5 is a photograph of an ethidium bromide stained agarose gel of a sample of PCR amplification of Dra DNA polymerase I under different reaction buffer conditions in the presence or absence of the indicated concentrations of betaine, MMNO or proline. . FIG. 6 shows p53 under different reaction buffer conditions (Mg ⁺⁺ concentration) in the presence or absence of a mixture of MMNO and proline in different ratios, or in the presence of betaine, MMNO or proline alone. It is a photograph of an ethidium bromide stained agarose gel of a sample of PCR amplification of exon 10. FIG. 7 shows Dra under different reaction buffer conditions (Mg ⁺⁺ concentration) in the presence or absence of a mixture of MMNO and proline in different ratios, or in the presence of betaine, MMNO or proline alone. It is a photograph of an ethidium bromide stained agarose gel of a sample of PCR amplification of DNA polymerase I. FIG. 8 is a photograph of an ethidium bromide stained agarose gel of a sample of PCR enrichment of a GC-rich P32D9 template, which shows the effect of a mixture of MMNO and proline, or betaine on the optimal annealing temperature. FIG. 9 shows the ethidium bromide sample of PCR amplification of the fragile X locus from the genomic DNA of the K562 cell line in the presence of various concentrations of either betaine or a 1: 1 mixture of MMNO and proline. It is a photograph of a stained agarose gel. Lane 1: no co-solvent; Lane 2: 0.25M; Lane 3: 0.5M; Lane 4: 0.75M, Lane 5: 1M, Lane 6: 1.25M, Lane 7: 1.5M, Lane 8: 1.75M, Lane 9: 2M. M: DNA size marker. FIG. 10 is a photograph of an ethidium bromide stained agarose gel of samples of PCR amplification of two different long GC-rich adenovirus DNA fragments in the presence or absence of a 1: 1 mixture of MMNO and proline at different concentrations. It is. Lane 1: no co-solvent; Lane 2: 0.25M; Lane 3: 0.5M; Lane 4: 1.0M. M: DNA size marker. FIG. 11 shows the presence or absence of various concentrations of 1: 1 mixture of MMNO and proline (lane A), betaine (lane B), L-carnitine (lane C), or DL-pipecolic acid (lane D). FIG. 5 is a photograph of an ethidium bromide stained agarose gel of a sample of PCR amplification of a GC-rich fragment of K562 genomic DNA in the presence. Lane 1: no co-solvent; Lane 2: 0.25M; Lane 3: 0.5M; Lane 4: 1M; Lane 5: 1.5M; Lane 6: 2M. M: DNA size marker. FIG. 12 shows an ethidium bromide stained agarose gel of a sample of PCR amplification of a GC-rich fragment of K562 genomic DNA in the presence or absence of various concentrations of betaine (lane A) or ectoin (lane B). It is a photograph of. Lane 1: no co-solvent; Lane 2: 0.25M; Lane 3: 0.5M; Lane 4: 1M; Lane 5: 1.5M; Lane 6: 2M. M: DNA size marker. FIG. 13 shows the structures of a number of exemplary compounds that can be used in accordance with the present invention.

(Detailed description of the invention)
(Definition)
In the following description, a number of terms used in recombinant DNA technology are utilized extensively. In order to provide a clearer and more consistent understanding of the detailed description and claims, including the scope to be given by such terms, the following definitions are provided.

  Library. As used herein, the term “library” or “nucleic acid library” refers to a set of nucleic acid molecules (circular or linear) that represent all or a significant portion of the DNA content of one organism. ) ("Genomic library") or a set of nucleic acid molecules ("cDNA library") that represent all or a significant portion of a gene expressed in a cell, tissue, organ or organism. Such libraries may or may not be included in one or more vectors.

  vector. As used herein, a “vector” is a plasmid, cosmid, phagemid, or phage DNA or other DNA molecule that can replicate autonomously in a host and recognize one or a few restriction endonucleases. It is characterized by the site. At this recognition site, such a DNA sequence can be cleaved in a deterministic manner without losing the essential biological function of the vector, and into which DNA is inserted to replicate and clone it. Can be made. The vector can further include a marker suitable for use in identifying cells transformed with the vector. For example, markers include, but are not limited to, tetracycline resistance or ampicillin resistance.

  Primer. As used herein, a “primer” refers to a single-stranded oligonucleotide that is extended by covalent attachment of nucleotide monomers during replication or polymerization of a DNA molecule.

  template. The term “template” as used herein refers to a double-stranded or single-stranded nucleic acid molecule that is to be amplified, synthesized, or sequenced. In the case of double-stranded molecules, denaturing the strands to form first and second strands is preferably done before these molecules can be amplified, synthesized, or sequenced. Alternatively, double-stranded molecules can be used directly as templates. For a single-stranded template, a primer that is complementary to a portion of the template hybridizes under appropriate conditions, and then one or more polymerases synthesize nucleic acid molecules that are complementary to all or part of the template. obtain. Alternatively, for a double-stranded template, one or more promoters (eg, SP6 promoter, T7 promoter or T3 promoter) are used in combination with one or more polymerases to complement all or part of the template. Nucleic acid molecules can be made. In accordance with the present invention, a newly synthesized molecule can be equal in length or shorter than the original template.

  Capture. The term “incorporation” as used herein means to be part of a DNA molecule and / or RNA molecule or primer.

  amplification. As used herein, “amplification” refers to any in vitro method for increasing the copy number of a nucleotide sequence through the use of a polymerase. Nucleic acid amplification results in the incorporation of nucleotides into DNA and / or RNA molecules or primers, thereby forming new molecules that are complementary to the template. The formed nucleic acid molecule and its template can be used as a template to synthesize additional nucleic acid molecules. As used herein, an amplification reaction can consist of multiple replicates. Examples of the DNA amplification reaction include polymerase chain reaction (PCR). One PCR reaction can consist of denaturation and synthesis of 5-100 “cycles” of DNA molecules.

  Oligonucleotide. An “oligonucleotide” is a covalently linked sequence of nucleotides joined by a phosphodiester bond between the 3 ′ position of a deoxyribose or ribose of one nucleotide and the 5 ′ position of a deoxyribose or ribose of an adjacent nucleotide. Synthetic or natural molecules, including. nucleotide. As used herein, “nucleotide” refers to a base-sugar-phosphate combination. Nucleotides are monomeric units (DNA and RNA) of nucleic acid sequences. The term nucleotide includes ribonucleoside triphosphates (ATP, UTP, CTP, GTP and deoxyribonucleoside triphosphates such as dATP, dCTP, dITP, dUTP, dGTP, dTTP or derivatives thereof). Derivatives include, for example, [αS] dATP, 7-deaza-dGTP and 7-deaza-dATP and nucleotide derivatives that confer nuclease resistance to nucleic acid molecules containing them. Where used, dideoxyribonucleoside triphosphates (ddNTPs) and derivatives thereof, illustrative examples of dideoxyribonucleoside triphosphates include ddATP, ddCTP, ddGTP, ddITP, and ddTTP. In accordance with the present invention, the “nucleotide” may be unlabeled or detectably labeled by well-known techniques, including, for example, radioisotopes, fluorescent labels , Chemiluminescent labels, bioluminescent labels and enzyme labels.

  Hybridization. The terms “hybridization” and “hybridize” refer to the base pairing of two complementary single-stranded nucleic acid molecules (RNA and / or DNA) to give a double-stranded molecule. As used herein, two nucleic acid molecules can hybridize even if the base pairs are not perfectly complementary. Thus, mismatched bases do not interfere with the hybridization of two nucleic acid molecules when appropriate conditions well known in the art are used.

  unit. The term “unit” as used herein refers to the activity of an enzyme. For example, when referring to thermotolerant DNA polymerase, one unit of activity incorporates 10 nanomoles of dNTP into acid insoluble material (ie, DNA or RNA) under standard primed DNA synthesis conditions for 30 minutes. Refers to the amount of enzyme.

  Other terms used in the fields of recombinant DNA technology and molecular biology and cell biology as used herein are generally understood by those of ordinary skill in the applicable arts.

(Outline)
The present invention relates generally to compounds, compositions and methods useful in enhancing the synthesis of nucleic acid molecules, particularly GC-rich nucleic acid templates. In particular, the present invention provides a compound having a formula selected from the group consisting of Formula I and Formula II below and a composition comprising one or more compounds, or a salt or derivative thereof. Preferably, at least 2, such as at least 3, at least 4, at least 5, at least 6, such compounds or compositions are used according to the present invention. Most preferably, 2-6, 2-5, 2-4, or 2-3 such compounds or compositions are used. The compound or composition of the present invention may exist in the form of a salt.
Formula I

ここで、Ａは、

Where A is

であり、ここで、Ｘは、

Where X is

であり、ここでＺは、Ｙと同じであっても、異なっていてもよく、
ここで、ＹおよびＺは各々独立して、−ＯＨ、−ＮＨ₂、−ＳＨ、ＰＯ₃Ｈ、−ＣＯ₂Ｈ、−ＳＯ₃Ｈおよび水素からなる群より選択され、ｆは、０〜２の整数であり、ｍは０〜２０の整数であり、そしてｅは、０〜２の整数であり；
ここで、Ｒ₄、Ｒ₅およびＲ₆は、同一であっても、異なっていてもよく、そして独立して、水素、アルキル、アルケニル、アルキニル、アリール、アミノ、チオール、メルカプタン、ハロ、ニトロ、ニトリロ、ヒドロキシ、ヒドロキシアルキル、ヒドロキシアリール、ホスファト、アルコキシ、オキシド、エーテル、エステル（アルカノイルオキシ）、カルボキシ、カルボニル、スルホニル、スルホン基およびアミド基からなる群より選択され、そしてｄは、０〜２の整数であり；
ここで、ａ、ｂおよびｃは、独立して０〜１の整数であり、ただし、ａ、ｂおよびｃの２つ以下が０であり；
ここで、Ｒ₁、Ｒ₂およびＲ₃は、同じであっても、異なっていてもよく、そして独立して、以下からなる群より選択され：
ａ）＝Ｏ
ｂ）（Ｗ）_g
｜
−（ＣＲ₇）_n；
ここで、Ｒ₇およびＷは各々、同じであっても、異なっていてもよく、そして独立して、水素、アルキル、アルケニル、アルキニル、アリール、アミノ、チオール、メルカプタン、ハロ、ニトロ、ニトリロ、ヒドロキシ、ヒドロキシアルキル、ヒドロキシアリール、ホスファト、アルコキシ、オキシド、エーテル、エステル（アルカノイルオキシ）、カルボキシ、カルボニル、スルホニル、スルホン基およびアミド基からなる群より選択され；ｇは、０〜２の整数であり；そしてｎは０〜２０の整数であり；そして
ここで、ｑは、１〜１００，０００であり得る。

Where Z may be the same as or different from Y,
Here, Y and Z are each independently selected from the group consisting of —OH, —NH ₂ , —SH, PO ₃ H, —CO ₂ H, —SO ₃ H and hydrogen, and f is 0 to 2 M is an integer from 0 to 20 and e is an integer from 0 to 2;
Wherein R ₄ , R ₅ and R ₆ may be the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, thiol, mercaptan, halo, nitro, Selected from the group consisting of nitrilo, hydroxy, hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, carbonyl, sulfonyl, sulfone group and amide group, and d is 0-2 An integer;
Here, a, b and c are each independently an integer of 0 to 1, provided that two or less of a, b and c are 0;
Wherein R ₁ , R ₂ and R ₃ may be the same or different and are independently selected from the group consisting of:
a) = O
b) (W) _g
｜
-(CR ₇ ) _n ;
Where R ₇ and W may each be the same or different and are independently hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, thiol, mercaptan, halo, nitro, nitrilo, hydroxy , Hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, carbonyl, sulfonyl, sulfone group and amide group; g is an integer from 0 to 2; And n is an integer from 0 to 20; and where q can be from 1 to 100,000.

  In the compound of formula I, when q = 1, the compound of formula I can be considered a monomer, and when q = 2-100,000, the compound of formula I is 2-100,000 monomers Can be considered a multimer or polymer composed of The monomers can each have the same structure or different structures, and can be linked by one or more bonds through one or more groups to form multimers of compounds of formula I (eg, Polymer).

  In a preferred aspect, when a, b or c is 0, the corresponding R group is an electron pair.

In another preferred embodiment, q = 1 and (R ₁ ) _a , (R ₂ ) _b and (R ₃ ) _c are ═O and the other two R groups are the same or If different and independently selected from the group consisting of hydrogen, methyl, ethyl and propyl, A is not methyl, ethyl or propyl.
Formula II

ここで、式ＩＩは、飽和であるか、または不飽和であり；
ここで、ｑは、１〜１００，０００であり得；
ここで、Ｘは、Ｎ、Ｃ、Ｏ、ＰおよびＳからなる群より選択され；
Ｙは、Ｏ、Ｎ、Ｓ、Ｐ、Ｃ、−Ｏ−ＮＨ−、−Ｏ−ＣＨ₂−Ｏ−、−Ｏ−Ｓ−、−Ｏ−ＣＨ₂−Ｓ−、−Ｏ−ＣＨ₂−ＮＨ−、−ＮＨ−Ｓ−、−ＮＨ−ＣＨ₂−ＮＨ−、−Ｏ−ＣＨ（ＣＨ₃）−ＮＨ−、−ＮＨ−ＣＨ（ＣＨ₃）−ＮＨ−、−Ｏ−ＣＨ（ＣＨ₃）−Ｏ−、−ＮＨ−Ｃ（ＣＨ₃）₂−ＮＨ−、−ＮＨ−ＣＨ₂−Ｓ−、および他のメルカプタン、ホスファト、アルコキシ、オキシド、エーテル、エステル（アルカノイルオキシ）、カルボキシ、スルホニル、スルホン基、およびアミド基からなる群より選択され；そして
ここで、Ｒ₁、Ｒ₂、Ｒ₃、Ｒ₄、Ｒ₅、Ｒ₆、Ｒ₇およびＲ₈は、同一であっても、異なっていてもよく、そして独立して、水素、アルキル、アルケニル、アルキニル、アリール、アミノ、チオール、メルカプタン、ハロ、ニトロ、ニトリロ、ヒドロキシ、ヒドロキシアルキル、ヒドロキシアリール、ホスファト、アルコキシ、オキシド、エーテル、エステル（アルカノイルオキシ）、カルボキシ、スルホニル、スルホン基およびアミド基からなる群より選択され；
ここで、ａ、ｂ、ｃ、ｄ、ｅ、ｍ、ｎ、およびｏは、整数であって、同じであっても、異なっていてもよく、そして独立して、ａ、ｂ、ｃ、ｄおよびｅについては、０〜２から選択され、そしてｍ、ｎおよびｏについては、０〜５から選択される。

Wherein formula II is saturated or unsaturated;
Where q can be 1 to 100,000;
Where X is selected from the group consisting of N, C, O, P and S;
Y is, O, N, S, P , C, -O-NH -, - O-CH 2 -O -, - O-S -, - O-CH 2 -S -, - O-CH 2 -NH -, - NH-S -, - NH-CH 2 -NH -, - O-CH (CH 3) -NH -, - NH-CH (CH 3) -NH -, - O-CH (CH 3) - O -, - NH-C ( CH 3) 2 -NH -, - NH-CH 2 -S-, and other mercaptans, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, sulfonyl, sulfone group And R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ and R ₈ may be the same or different from each other. Well and independently hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, thiol, mercap Emissions, halo, nitro, nitrilo, hydroxy, hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, sulfonyl, selected from the group consisting of sulfone and amide groups;
Where a, b, c, d, e, m, n, and o are integers that may be the same or different and are independently a, b, c, d And e are selected from 0 to 2, and m, n and o are selected from 0 to 5.

  In the compound of formula II, when q = 1, the compound of formula II can be considered a monomer, and when q = 2-100,000, the compound of formula II is 2-100,000 monomers Can be considered a multimer or polymer composed of The monomers may each have the same structure or different structures, and are linked by one or more bonds through one or more groups to form a multimer of compounds of formula II (eg, Polymer).

  In one preferred aspect of the invention, Y and / or Z is N and m, n and o are 1. In another preferred aspect, Y and / or X is N and / or O, and m and n are 1, and o is 2. Preferably, when a, b, c, d and / or e is 0, the corresponding R group is an electron pair or is involved in the formation of an unsaturated structure.

For compounds of Formula I and Formula II:
Representative C _6-14 aryl groups include but are not limited to: phenyl group, benzyl group, methylindolyl group, naphthyl group, phenanthryl group, anthracyl group, indenyl group, azulenyl group, biphenyl group, biphenylenyl A group, and a fluorenyl group;
Exemplary halo groups include but are not limited to: fluorine, chlorine, bromine and iodine;
Representative C _1-15 alkyl groups include, but are not limited to: methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl , A decyl group, and a branched chain alkyl group;
Exemplary C _2-15 alkenyl groups include, but are not limited to: ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, and the like, and A branched alkenyl group;
Exemplary C _2-15 alkynyl groups include: ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, and the like, as well as branched alkynyl groups ;
Exemplary lower alkoxy (ether) groups include oxygen substitutions with one of the C _1-4 alkyl groups referred to above; and exemplary C _2-6 alkanoyloxy groups include acetoxy, propionyloxy, butane Including noyloxy, pentanoyloxy, hexanoyloxy and their branched isomers.

  Compounds that can be used according to the present invention include sugars, amino acids and polyhydric alcohols and their derivatives. Examples of saccharides are oligosaccharides and monosaccharides (e.g. trehalose, maltose, glucose, sucrose, lactose, xylobiose, agarobiose, cellobiose, levanbiose, chitobiose, 2-β-glucuronosyl glucuronic acid ( 2-β-glucuronosylglucuronic acid), allose, altrose, galactose, gulose, idose, mannose, talose, sorbitol, levulose, xylitol and arabitol).

  Such amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine, and those Derivatives thereof, but are not limited thereto. Both D and L amino acids as well as non-protein amino acids can be used according to the present invention. Examples include N- (3′-one-5′-methyl) -hexylalanine, leucine betaine, N-methylisoleucine, and γ-glutamylleucine.

  Examples of polyhydric alcohols include, but are not limited to, glycerol, ethylene glycol, polyethylene glycol, and the like.

Preferred compounds of the present invention include 4-methylmorpholine N-oxide (MMNO), and N-alkylimidazole compounds such as 1-methylimidazole, 2-methylimidazole, and 4-methylimidazole, betaine (carboxymethyl-trimethylammonium ), Taurine, ectoine, pipecolic acid, pipecolic acid, 2-morpholinoethanesulfonic acid, pyridine N-oxide, N, N-dimethyloctylamine N-oxide, 3-methylisoxazole-5 (4H) ) -One morpholine salt, glycine, sorcosine, NN-dimethylglycine, N-methyl-proline, 4-hydroxy-proline, 1-methyl-2-pyrrolecarboxylic acid, 1-methylindole -2-carboxylic acid, 2-pyrazinecarboxylic acid, 5-methyl-2-pyrazinecarboxylic acid, 4-methyl-5-imidazole-carboxaldehyde, 1-methylpyrrole-2-carboxylic acid, 1-ethyl nitrate -3-methylimidazolium, ethylazetidine-1-propionate, N, N-dimethyl-phenylalanine, S-carboxymethyl-cysteine, 2-imidazolecarboxaldehyde, 4-imidazoleacetic acid, 4-imidazole carboxylic acid, 4,5-imidazole dicarboxylic acid (imidazdedicarboxylic acid), carnitine N ^e - acetyl -b- lysine, .gamma.-aminobutyric acid, Lance-4-hydroxystachydrin, Nα-carbamoyl-L-glutamine l-amide, choline, dimethylthetin, (sulfobetaine and dimethylacetetine and their derivatives), N-acetylglutaminylglutamineamide, dimethylsulfonio Propionate (dimethylsulfoniopropionate), ectoine (1,4,5,6-tetrahydro-2methyl-4-pyrimidine carboxylic acid), hydroxyectoine, glutamic acid, β-glutamine, octopine, sarcosine, and trimethylamine N-oxide (TMAO), poly (2-ethyl-2-oxazoline) having an average molecular weight of about 50,000 to about 500,000 daltons, average molecular weight Poly about 100,000 to about 200,000 Daltons may include (diallyl dimethyl ammonium chloride) and other amino acids and derivatives thereof, but is not limited thereto.

Further preferred compounds include derivatives and salts of compounds of formulas I and II. For example, when a compound of formula I or formula II contains a carboxyl (C═O) group, the compounds of the invention can be synthesized by conventional methods of chemical synthesis (eg, aggregating a carboxyl-containing compound and an alcohol or amino compound). Including esters and amides of carboxyl groups that can be prepared using Examples of useful alcohols according to this aspect of the invention include C _1-6 alcohols and C _7-12 aralkanol compounds, which are methanol, ethanol, propanol, butanol, pentanol, hexanol, and Including but not limited to those branched chain isomers. Examples of useful amino compounds according to this aspect of the invention include C _1-6 amino compounds and C _7-12 aralkamino compounds, which are methylamine, ethylamine, propylamine, butylamine, pentylamine, Including but not limited to hexylamine and their branched isomers. Where the compound of formula I or formula II contains a hydroxy (—OH) group, the compound of the invention can be combined with a hydroxy-containing compound such as C _1-6 alkanoic acid, C _6-12 aralkanoic acid or C _2-12 dialkanoic acid or its anhydride (for example, formic acid, acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid and their branched chain isomers, and succinic acid, succinic anhydride, fumaric acid, maleic acid, etc. And esters of such compounds, which can be prepared by aggregation. Other derivatives of the compounds of Formula I and Formula II that can be prepared and used in accordance with the present invention will be apparent to those skilled in the art in view of the teachings contained herein and knowledge in the art.

  Also included within the scope of the invention are salts of the compounds of Formulas I and II. Acid-added salts of compounds of formulas I and II can be prepared by conventional chemical synthesis methods (eg hydrochloric acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, tartaric acid, carboxylic acid, phosphoric acid, oxalic acid By mixing a particular compound solution with a solution of an acid containing, etc. The basic salts of the compounds of formulas I and II can be prepared by conventional methods of chemical synthesis (for example, with certain basic solutions such as sodium hydroxide, potassium hydroxide, choline hydroxide, sodium carbonate, Tris, etc.). By mixing compound solutions). Other salts of the compounds of formulas I and II that can be prepared and used in accordance with the present invention will be apparent to those skilled in the art in view of the teachings contained herein and knowledge in the art.

  The above compounds and compositions can be used alone or in any combination thereof. Preferably, at least two combinations, at least three combinations, at least four combinations, at least five combinations and the like are used according to the present invention. In a preferred aspect, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, and 2-3 such compounds are ,used. In a preferred aspect, the present invention relates to a composition obtained by mixing any combination of the above compounds. When mixed with such compounds, certain interactions can occur, which can change one or more structures of the compound being mixed and can result in new or different compounds.

  These compositions can be used in methods for enhanced, high performance synthesis of nucleic acid molecules, including amplification methods (especially PCR), reverse transcription methods, and sequencing methods. The invention also relates to nucleic acid molecules produced by these methods, fragments or derivatives thereof, and vectors and host cells comprising such nucleic acid molecules, fragments or derivatives. The invention also relates to the use of such nucleic acid molecules to produce the desired polypeptide. The invention also relates to a kit comprising a compound or composition of the invention.

(Synthesis method)
Compounds of formula I and II can be synthesized using standard techniques of organic chemical synthesis known to those skilled in the art as follows.

  The synthesis of compounds of formula I can be carried out as follows:

例えば、ここでＲ₄およびＺはＨであり、Ｙは−ＣＯ₂Ｈであり、ｄ，ｅ、ｆ、およびｍは、１であり、次いで、出発化学物質は、市販のＢｒＣＨ₂ＣＨ₂ＣＯ₂Ｈである。

For example, where R ₄ and Z are H, Y is —CO ₂ H, d, e, f, and m are 1, then the starting chemical is commercially available BrCH ₂ CH ₂ CO ₂ H.

  The synthesis of the compound of formula II can be carried out as follows:

例えば、ここで、Ｒ₂およびＲ₃は、Ｈであり；ｂおよびｍは、１であり、；ｃ−１およびｎ−１は、０であり、次いで、出発化学物質は、市販のＮＨ₂−ＣＨ₂−ＣＯＨである。

For example, where R ₂ and R ₃ are H; b and m are 1; c-1 and n-1 are 0, then the starting chemical is commercially available NH ₂ it is a -CH ₂ -COH.

_{BrCH 2 CO 2 H, CH 3} CH (Br) CO 2 H, CH 2 CH 2 CH (Br) CO 2 H, BrCH 2 CH 2 CH 2 CO 2 H, Cl-CH 2 -CH 2 -Cl (CH 3 ) ₂ CHCH (Br) CO ₂ , CH ₃ CH ₂ CH (Br) CO ₂ H, BrCH ₂ CH ₂ CH ₂ CO ₂ H, BrCH ₂ CH ₂ CO ₂ H, HO ₂ CCH ₂ CH (Br) CO ₂ H Is also commercially available. Such compounds are available from Aldrich (St. Louis, MO).

  Numerous compounds for use in the present invention, such as amino acids and their derivatives, saccharides and their derivatives, and N-alkylimidazole compounds (including 1-methylimidazole and 4-methylimidazole) are: For example, commercially available from Sigma (St. Louis, MO).

  To formulate the composition of the present invention, the one or more compounds described above can be mixed together in any manner. Such mixtures include the steps of mixing these compounds in their powder form, preparing a solution of each compound in an aqueous or organic solvent, as well as forming a solution of the present invention. Or by preparing a solution of at least one compound and mixing one or more additional compound powder forms.

  In a further preferred aspect of the present invention, the composition of the present invention may further comprise one or more polypeptides having nucleic acid polymerase activity. Such preferred enzymes having nucleic acid polymerase activity may include, but are not limited to, polypeptides having DNA polymerase activity, polypeptides having RNA polymerase activity, and polypeptides having reverse transcriptase activity.

  More preferably, the composition of the present invention is provided at a working concentration or as a concentrate (2 ×, 5 ×, 10 ×, 50 ×, etc.). Such compositions are preferably stable upon storage at various temperatures. The terms “stable” and “stability” as used herein generally indicate that the composition is about 1 week at a temperature of about 4 ° C., about 6 months at a temperature of about −20 ° C. Retain at least 70%, preferably at least 80%, and most preferably about 90% of the activity of the original enzyme and / or compound (eg, the compound or enzyme of the composition) after storage. means. As used herein, the term “working concentration” refers to the concentration of a chemical compound or enzyme that is at or near the optimal concentration used in solution to perform a particular function (eg, nucleic acid synthesis). Means.

  The water that can be used in forming the compositions of the present invention is preferably distilled, deionized, and sterile filtered (through a 0.1-0.2 μm filter) and contaminated with DNase and RNase enzymes. There is no water. Such water is commercially available, for example, from Sigma Chemical Company (Saint Louis, Missouri) or, if necessary, can be made according to methods well known to those skilled in the art.

  In addition to chemical (and optionally polypeptide) components, the compositions of the invention preferably include one or more buffers and cofactors necessary for the synthesis of the nucleic acid molecule. Particularly preferred buffers for use in forming the compositions of the present invention are acetate, sulfate, hydrochloride, phosphate, or the free acid of tris- (hydroxymethyl) aminomethane (TRIS®). An alternative buffer of approximately the same ionic strength and pKa can be used as TRIS®, which is in form but with equivalent results. In addition to the buffer salts, cofactor salts such as potassium salts (preferably potassium chloride or potassium acetate) and magnesium salts (preferably magnesium chloride or magnesium acetate) are included in the composition.

  Often, it is preferred to first dissolve the buffer and cofactor salt in water at a working concentration and adjust the pH of the solution before adding the chemical compound (and optionally the polypeptide). In this way, any pH sensitive chemical compounds and polypeptides are less susceptible to acid mediated or alkali mediated inactivation or degradation during formulation of the compositions of the invention.

  To formulate a buffered salt solution, preferably a salt of tris (hydroxymethyl) aminomethane (TRIS®) and most preferably a salt of the buffer which is a salt of its hydrochloride is Combined with a sufficient amount of water, a concentration of 5 to 150 millimolar TRIS®, preferably 10 to 60 millimolar TRIS®, most preferably about 20 to 60 millimolar TRIS® Resulting in a solution having the trademark). Magnesium salt (preferably either its chloride or acetate) is added to this solution and 1-10 millimolar, preferably 1.5-8.0 millimolar, and most preferably about 3-7.5. It can provide that working concentration of millimolar. A potassium salt (most preferably potassium chloride) may also be added to the solution at a working concentration of 10-100 millimolar and most preferably at a working concentration of about 75 millimolar. A reducing agent such as dithiothreitol is preferably at a final concentration of about 1-100 mM, more preferably at a concentration of about 5-50 mM or about 7.5-20 mM, and most preferably at a concentration of about 10 mM. Can be added to the solution. A small amount of EDTA salt such as disodium ethylenediaminetetraacetic acid (EDTA) may also be added (preferably about 0.1 mM), but the inclusion of EDTA is essential for the function or stability of the composition of the invention. There seems to be no. After adding all buffers and salts, the buffered salt solution is mixed well until all salts are dissolved and the pH is 7.4-9. Using methods known in the art. PH value of 2, preferably from 8.0 to 9.0, and most preferably at a pH value of about 8.4.

  To these buffered salt solutions, the compounds of the invention and optionally one or more polypeptides having nucleic acid polymerase activity are added to produce the compositions of the invention.

  In preferred compositions, the compounds of the present invention are about 10: 1, about 9: 1, about 8: 1, about 7: 1, about 6: 1, about 5: 1, about 4: 1, about 3: 1. About 2.5: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, about 1: 1, about 1: 1.25, about 1: 1. .5, about 1: 1.75, about 1: 2, about 1: 2.5, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about 1: 7, about 1: 8, about 1: 9, or about 1:10 molar ratio or stoichiometric ratio. More preferably, the compounds are mixed in a molar ratio or stoichiometric ratio of about 1: 1. Other molar ratios or stoichiometric ratios can be determined by routine optimization. When more than two compounds are used to form the composition of the invention, the amount of each compound can be easily optimized by testing its effect on nucleic acid synthesis. These compounds are then used in the nucleic acid synthesis methods described below for about 0.01-5M, about 0.05-5M, about 0.1-4M, about 0.25-3M, about 0. Preferably formulated in the composition at a working concentration of 3 to 2.5M, about 0.4 to 2M, about 0.4 to 1.5M, about 0.4 to 1M, or about 0.4 to 0.8M. Is done. Depending on the compound used, other molar amounts can be used depending on the desired result. The composition of the invention is then allowed to use at 65 ° C. for 2-4 weeks, room temperature to 37 ° C. for 1-2 months, 4 ° C. for 1-6 months, and −20 ° C. for 3 months until used for the synthesis of nucleic acid molecules. It can be stored from months to over a year.

Various polypeptides having polymerase activity are useful according to the present invention. Enzymes such as nucleic acid polymerases (including DNA polymerase and RNA polymerase) are included in these polypeptides. Such polymerases include, but are not limited to, Thermus thermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNA polymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotoga maritima co (Tma) DNA polymerase, or VENT ^TM) DNA polymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT ^TM DNA polymerase, Pyrococcus woosii (Pwo) DNA polymerase, Pyrococcus sp KDD2 (KOD) DNA polymerase, Bacillus terothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNA polymerase, Sulfolobus acidocaldarius (Sac) DNA polymerase, Thermoplasma acidophilum (Tac) DNA polymerase, Thermosflavus (Tac) (DYNAZYME ^™ ) DNA polymerase, Methanobacterium thermoautotropicum (Mth) DNA polymerase, mycobacterium DNA polymerase (Mtb, Mlep), and their Mutants, variants and derivatives. RNA polymerases such as T3, T5 and SP6, and variants, variants and derivatives thereof can also be used according to the present invention.

The nucleic acid polymerase used in the present invention can be mesophilic or thermophilic and is preferably thermophilic. Preferred mesophilic DNA polymerases include T7 DNA polymerase, T5 DNA polymerase, Klenow fragment DNA polymerase, DNA polymerase III, and the like. Preferred thermostable DNA polymerases that can be used in the methods and compositions of the present invention are Taq, Tne, Tma, Pfu, Tfl, Tth, Stoffel fragments, VENT ^™ and DEEPVENT ^™ DNA polymerases, and variants, variants thereof And derivatives (US Pat. No. 5,436,149; US Pat. No. 4,889,818; US Pat. No. 4,965,188; US Pat. No. 5,079,352; US Pat. US Pat. No. 5,374,553; US Pat. No. 5,270,179; US Pat. No. 5,047,342; US Pat. No. 5,512,462; WO 92/06188; WO 92/06200; WO 96/10640; Barnes, WM, Gene 112: 29-35 (1 92); Lawyer, FC et al., PCR Meth. Appl. 2: 275-287 (1993); Flaman, J.-M et al., Nucl. Acids Res. 22 (15): 3259-3260 (1994)). . For amplification of long nucleic acid molecules (eg, nucleic acid molecules longer than about 3-5 Kb), at least two DNA polymerases (one substantially lacking 3 'exonuclease activity and one is 3' Having exonuclease activity) is typically used. U.S. Pat. No. 5,436,149; U.S. Pat. No. 5,512,462; Barnes, W. et al. M.M. Gene 112: 29-35 (1992); and co-pending US patent application Ser. No. 08 / 801,720 (filed Feb. 14, 1997), the entire disclosures of which are incorporated herein by reference. See). Examples of DNA polymerases that substantially lack 3 ′ exonuclease activity are Taq, Tne (exo ⁻ ), Tma (exo ⁻ ), Pfu (exo ⁻ ), Pwo (exo ⁻ ), and Tth DNA polymerases, and their mutations Including but not limited to body variants and derivatives.

Polypeptides having reverse transcription activity for use in the present invention include any polypeptide having reverse transcription activity. Such enzymes include retrovirus reverse transcriptase, retrotransposon reverse transcriptase, hepatitis B reverse transcriptase, cauliflower mosaic virus reverse transcriptase, bacterial reverse transcriptase, Tth DNA polymerase, Taq DNA polymerase (Saiki, R. et al. K. et al., Science 239: 487-491 (1988); U.S. Pat. Nos. 4,889,818 and 4,965,188), Tne DNA polymerase (WO96 / 10640), Tma DNA polymerase (U.S. Pat. 5,374,553), and variants, modifications, and derivatives thereof (eg, co-pending US patent application Ser. Nos. 08 / 706,702 and 08 of A. John Hughes and Deb K. Chatterjee). / 706,706 (both 1 Including, but not limited to, filed September 9, 996), which are hereby incorporated by reference in their entirety. Preferred enzymes for use in the present invention include enzymes that decrease or substantially reduce RNase H activity. An enzyme that “substantially decreases in RNase H activity” means that the enzyme corresponds to the corresponding wild type or RNase H ⁺ enzyme (eg, wild type Moloney murine leukemia virus (M-MLV) reverse transcriptase, avian myeloblastosis Less than about 20%, more preferably less than about 15%, 10%, or 5%, and most preferably about 2 of the RNase H activity of a virus (AMV) reverse transcriptase, or Rous sarcoma virus (RSV) reverse transcriptase) Means having less than% activity. The RNase enzyme activity of any enzyme is determined by various assays (eg, US Pat. No. 5,244,797, Kotewicz, ML, et al., Nucl. Acids Res. 16: 265 (1988) and Gerard, G. F.). Et al, FOCUS 14 (5): 91 (1992)), the entire disclosure of which is hereby incorporated by reference in its entirety. Particularly preferred such polypeptides for use in the present invention, M-MLV H ^- reverse transcriptase, RSV H ^- reverse transcriptase, AMV H ^- reverse transcriptase, RAV (rous-associated virus) H ^- reverse transcriptase , MAV (myeloblastosis-associated virus) H ^- reverse transcriptase and HIV H ^- including reverse transcriptase, but not limited to. However, any enzyme capable of generating a DNA molecule from a ribonucleic acid molecule that has substantially reduced RNase H activity (ie, having reverse transcriptase activity) is not included in the compositions, methods and kits of the present invention. It will be appreciated by those skilled in the art that

  DNA polymerases and RNA polymerases for use in the present invention are described, for example, in Life Technologies, Inc. (Rockville, Maryland), Perkin-Elmer (Branchburg, New Jersey), New England BioLabs (Beverly, Massachusetts) or commercially available from Boehringer Mannheim Biochem. Polypeptides with reverse transcription activity for use in the present invention are described in, for example, Life Technologies, Inc. (Rockville, Maryland), Pharmacia (Piscataway, New Jersey), Sigma (Saint Louis, Missouri) or Boehringer Mannheim Biochemicals (obtained commercially from Indianapolis, Indianapolis). Alternatively, polypeptides having reverse transcription activity can be isolated from their natural viral or bacterial sources according to standard procedures for isolating and purifying natural proteins well known to those skilled in the art (eg, Houts, DE et al., J. Virol. 29: 517 (1979)). In addition, polypeptides having reverse transcription activity can be prepared by recombinant DNA techniques known to those of skill in the art (eg, Kotewicz, ML et al., Nucl. Acids Res. 16: 265 (1988); Soltis, D A. and Skalka AM, Proc. Natl. Acad. Sci. USA 85: 3372-3376 (1988)).

  The polypeptide having polymerase activity or reverse transcriptase activity is preferably about 0.1-200 units / ml, about 0.1-50 units / ml, about 0.1-40 units / ml, about 0.1 In a solution of about -36 units / ml, about 0.1-34 units / ml, about 0.1-32 units / ml, about 0.1-30 units / ml, or about 0.1-20 units / ml Used in the compositions and methods of the present invention at a final concentration, and most preferably at a concentration of about 20-40 units / ml. Of course, other suitable concentrations of such polymerases or reverse transcriptases suitable for use in the present invention will be apparent to those skilled in the art.

(Method of nucleic acid synthesis)
The compounds and compositions of the invention can be used in methods for the synthesis of nucleic acids. In particular, the compounds and compositions of the present invention provide for the synthesis of nucleic acid molecules having a high content of guanine and cytosine (ie, “GC-rich” nucleic acid molecules), particularly through amplification reactions such as the polymerase chain reaction (PCR). Has been found to facilitate. Thus, the compounds and compositions of the invention can be used in any method that requires the synthesis of nucleic acid molecules, such as DNA (particularly cDNA) and RNA (particularly mRNA) molecules. Methods in which the compounds or compositions of the invention can be advantageously used include, but are not limited to, nucleic acid synthesis methods, nucleic acid amplification methods, nucleic acid reverse transcriptases, and nucleic acid sequencing methods.

(Synthesis)
The nucleic acid synthesis method according to this aspect of the invention may include one or more steps. For example, the present invention includes (a) mixing a nucleic acid template with one or more of the above compounds and compositions of the present invention to form a mixture; and (b) complementary to all or part of the template. A method is provided for synthesizing a nucleic acid molecule comprising incubating the mixture under conditions sufficient to produce a first nucleic acid molecule. In accordance with this aspect of the invention, the nucleic acid template can be a DNA molecule (eg, a cDNA molecule or library), or an RNA molecule (eg, an mRNA molecule).

  In accordance with the present invention, an input nucleic acid molecule or library can be prepared from a population of nucleic acid molecules obtained from a natural source (eg, various cells, tissues, organs or organisms). Cells that can be used as a source of nucleic acid molecules include prokaryotes (bacterial cells, including cells of the following genus species; Escherichia, Bacillus, Serratia, Salmonella, Staphylococcus, Streptococcus, Clostridium, Chlamydia, Nemisper, Borrelia, Legionella, Pseudomonas, Mycobacterium, Helicobacter, Erwinia, Agrobacterium, Rhizobium, and Streptomyces, and eukaryotes (including: fungi (especially yeast), plants, protists, and , Drosoph la species cells), nematodes (particularly Caenorhabditis elegans cells), and mammals (particularly, be an animal), including human cells).

  Mammalian somatic cells that can be used as a source of nucleic acid molecules or a library of nucleic acid molecules include: blood cells (reticulocytes and leukocytes), endothelial cells, epithelial cells, nerve cells (central or peripheral nervous system) Origin), muscle cells (including muscle cells and myoblasts from skeletal muscle, smooth muscle, or heart muscle), connective tissue cells (fibroblasts, adipocytes, chondrocytes, chondroblasts, bone cells and osteoblasts) And other stromal cells (eg, macrophages, dendritic cells, Schwann cells). Mammalian germ cells (sperm cells and oocytes) can also be used as a source or library of nucleic acids for use in the present invention. Progenitors, precursors and stem cells that give rise to the above somatic and germ cells can be used as well. Mammalian tissues or organs are also suitable for use as a source of nucleic acids; brain tissue, kidney tissue, liver tissue, pancreas tissue, blood tissue, bone marrow tissue, muscle tissue, nerve tissue, skin Tissue, urogenital tissue, circulatory tissue, lymphoid tissue, gastrointestinal tissue and connective tissue sources, and tissues or organs from mammalian (including human) embryos or fetuses.

  Any prokaryotic or eukaryotic cell, tissue, and organ described above may be normal, pathological, transformant, tree solid, progenitor, precursor, fetal, or embryo Can be sex. Pathological cells can be, for example, infectious diseases (caused by bacteria, fungi or yeast, viruses (including HIV) or parasites), genetic or biochemical pathologies (eg, cystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy or multiple sclerosis), or cells involved in cancerous processes. Examples of transformed animal cell lines or established animal cell lines include COS cells, CHO cells, VERO cells, BHK cells, HeLa cells, HepG2 cells, K562 cells, and F9 cells. Other cells, cell lines, tissues, organs and organisms suitable as a nucleic acid source for use in the methods of the invention will be apparent to those skilled in the art. These cells, tissues, organs and organisms can be obtained from their natural sources or from sources such as the American Type Culture Collection (Rockville, Maryland) and other sources known to those skilled in the art. It can be obtained commercially.

  Once a starting cell, starting tissue, starting organ, or other starting sample is obtained, nucleic acid molecules (eg, DNA, RNA (eg, mRNA or polyA + RNA) molecules) can be isolated from or from cDNA molecules or cDNA libraries can be obtained by methods well known in the art (eg, Maniatis, T. et al., Cell 15: 687-701 (1978); Okayama, H. and Berg, P., Mol. Cell. Biol. 2). 161-170 (1982); Gubler, U. and Hoffman, BJ, Gene 25: 263-269 (1983)).

  In the practice of this aspect of the invention, the first nucleic acid molecule is a nucleic acid template (preferably a DNA molecule (eg, cDNA molecule), or RNA molecule (eg, mRNA molecule or poly A + RNA molecule) obtained as described above. ) Can be synthesized by mixing with one or more of the compounds or compositions of the present invention to form a mixture. Synthesis of a first nucleic acid molecule complementary to all or part of the nucleic acid template is achieved under conditions convenient for reverse transcription (in the case of RNA templates) and / or polymerization of the input nucleic acid molecule. Is done. Such synthesis is usually accomplished in the presence of nucleotides (eg, deoxyribonucleoside triphosphate (dNTP), dideoxyribonucleoside triphosphate (ddNTP), or derivatives thereof).

  Alternatively, the compounds, compositions and methods of the present invention can be used for the synthesis in a single tube of double stranded nucleic acid molecules. In this approach, the first nucleic acid molecule synthesized above is incubated under conditions sufficient to produce a second nucleic acid molecule that is complementary to all or part of the first nucleic acid molecule. This second strand synthesis can be achieved, for example, by a modified Gubler-Hoffman reaction (D'Alessio, JM, et al., Focus 9: 1 (1987)).

  Of course, other techniques of nucleic acid synthesis in which the compositions and methods of the invention can be advantageously utilized will be readily apparent to those skilled in the art.

(Amplification and sequencing methods)
In other aspects of the invention, the compositions of the invention can be used in methods for amplification or sequencing of nucleic acid molecules. The nucleic acid amplification method according to this aspect of the invention is a method of reversing in a method generally known in the art as a one-step (eg, one-step RT-PCR) or two-step (eg, two-step RT-PCR) reverse transcriptase amplification reaction. It may further include the use of one or more polypeptides having a coenzyme activity. For amplification of long nucleic acid molecules (ie, greater than about 3-5 Kb in length), the compositions of the present invention can be obtained from the same co-pending US application Ser. No. 08/08, filed Feb. 14, 1997. 801,720 (this disclosure may include combinations of polypeptides having DNA polymerase activity, as described in detail in this disclosure, the entirety of which is incorporated herein by reference.

  An amplification method according to this aspect of the invention may include one or more steps. For example, the invention includes (a) mixing a nucleic acid template with one or more of the compounds or compositions described above to form a mixture; and (b) complementary to all or part of the template. A method for amplifying a nucleic acid molecule is provided, comprising incubating the mixture under conditions sufficient to amplify the nucleic acid molecule. The invention also provides nucleic acid molecules amplified by such methods.

  General methods for amplification or analysis of nucleic acid molecules or fragments are well known to those skilled in the art (eg, US Pat. Nos. 4,683,195; 4,683,202; 800, 159; edited by Innis, MA, et al., PCR Protocols: A Guide to Methods and Applications, San Diego, California: Academic Press, Inc. (1990); Ed., PCR Technology: Current Innovations, Boca Raton, Florida: CRC Press (1994)). For example, amplification methods that can be used in accordance with the present invention include PCR (US Pat. Nos. 4,683,195 and 4,683,202), strand displacement amplification (SDA; US Pat. No. 5,455,166); EP 0684315), nucleic acid sequence-based amplification (NASBA; US Pat. No. 5,409,818; EP 0329822).

  Typically, these amplification methods include the following steps; a compound or composition comprising a nucleic acid sample in the presence of one or more primer sequences and comprising one or more polypeptides having nucleic acid polymerase activity (e.g. Contacting the compound or composition of the invention), amplifying the nucleic acid sample, preferably by PCR or equivalent automated amplification techniques, to produce a collection of amplified nucleic acid fragments, and optionally Analyzing the amplified nucleic acid fragment, preferably by gel electrophoresis, and for the presence of the nucleic acid fragment, for example by staining the gel with a nucleic acid staining dye such as ethidium bromide Process.

  After amplification by the method of the present invention, the amplified nucleic acid fragment can be isolated for further use or characterization. This step typically includes any physical or biochemical method (including gel electrophoresis, capillary electrophoresis, chromatography (including size chromatography, affinity chromatography, and immunochromatography), density gradient centrifugation and immunoadsorption. ) To separate the amplified nucleic acid fragments by size. Separation of nucleic acid fragments by gel electrophoresis is particularly preferred. Because it provides a rapid and highly reproducible and precise separation method for many nucleic acid fragments and allows direct, simultaneous comparison of fragments in several samples of nucleic acids . In another preferred embodiment, this approach can be expanded to isolate and characterize these fragments or any nucleic acid fragments amplified by the methods of the invention. Accordingly, the present invention also relates to an isolated nucleic acid molecule produced by the amplification or synthesis method of the present invention.

  In this embodiment, one or more amplified nucleic acid molecule fragments are removed from the gel used for identification (see above) according to standard techniques (eg, electroelution or physical excision). . The isolated unique nucleic acid fragment is then suitable for transfection or transformation of various prokaryotic (bacterial) cells or eukaryotic (yeast, plant, or animals including humans and other mammals) cells. It can be inserted into standard nucleotide vectors (including expression vectors). Alternatively, nucleic acid molecules amplified and isolated using the compounds, compositions and methods of the invention are described below, for example, by sequencing methods (ie, determining the nucleotide sequence of a nucleic acid fragment). And other methods standard in the art (see, eg, US Pat. Nos. 4,962,022 and 5,498,523 (which relate to DNA sequencing methods)) It can be further characterized.

  The nucleic acid sequencing method according to the present invention may comprise one or more steps. For example, the present invention provides a method for sequencing a nucleic acid molecule comprising: (a) a nucleic acid molecule to be sequenced comprising one or more primers, one or more of the above compounds and compositions of the present invention. Compound, one or more nucleotides, and one or more terminators (eg, dideoxynucleotides) to form a mixture; (b) a molecule complementary to all or part of the molecule to be sequenced Incubating the mixture under conditions sufficient to synthesize the population of; and (c) separating the population to determine the nucleotide sequence of all or part of the molecule to be sequenced Process.

  Nucleic acid sequencing techniques that can use the compositions of the present invention include dideoxy sequencing methods as disclosed in US Pat. Nos. 4,962,022 and 5,498,523.

(Vector and host cell)
The invention also relates to vectors containing the isolated nucleic acid molecules of the invention, host cells genetically engineered with recombinant vectors, and methods for producing recombinant polypeptides using these vectors and host cells.

  The vector used in the present invention can be, for example, a phage or a plasmid, and is preferably a plasmid. A vector containing a cis-acting control region for a nucleic acid encoding the polypeptide of interest is preferred. Appropriate trans-acting factors can be supplied by the host, supplied by a complementing vector, or supplied by the vector itself upon introduction into the host.

  In certain preferred embodiments in this regard, the vector provides for specific expression of the polypeptide encoded by the nucleic acid molecule of the present invention; such expression vector may be inducible and / or cell type specific. . Among such vectors, vectors derived from environmental factors that are easy to manipulate (eg, temperature and nutrient additives) are particularly preferred.

  Expression vectors useful in the present invention include chromosome-derived vectors, episome-derived vectors and virus-derived vectors (eg, vectors derived from bacterial plasmids or bacteriophages, and vectors derived from combinations thereof (eg, cosmids and phagemids)). ).

DNA insert, the phage λP _L promoter, E. Should be operably linked to a suitable promoter, such as the E. coli lac, trp, and tac promoters. Other suitable promoters are known to those skilled in the art. The gene fusion construct further includes a site for transcription initiation, termination, and a ribosome binding site for translation in the transcription region. The coding portion of the mature transcript expressed by the construct is preferably the translation start codon at the beginning of the polynucleotide to be translated and a stop codon (UAA, UGA or UAG) appropriately positioned at the end. including.

  The expression vector preferably includes at least one selectable marker. Such markers include E. coli. Examples include tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria.

  Particularly preferred vectors for use in the present invention include: pQE70, pQE60 and pQE-9 (available from Qiagen); pBS vector, Pagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (from Stratagene) PcDNA3 (available from Invitrogen); and pGEX, pTrxfus, pTrc99a, pET-5, pET-9, pKK223-3, pKK233-3, pRD540, pRIT5 (available from Pharmacia). Other suitable vectors will be readily apparent to those skilled in the art.

  Representative examples of suitable host cells include E. coli. bacterial cells such as, but not limited to, E. coli, Streptomyces species, Erwinia species, Klebsiella species, and Salmonella typhimurium. E. E. coli is preferred as the host cell and E. coli is preferred. E. coli strains DH10B and Stbl2 are particularly preferred. These are commercially available (Life Technologies, Inc; Rockville, Maryland).

(Peptide production)
As described above, the method of the present invention can be performed by inserting any of the above nucleic acid molecules or vectors into a host cell, and expressing any nucleotide sequence encoding the polypeptide of interest by the host cell. Suitable for production of polypeptides. Introducing a nucleic acid molecule or vector into a host cell to create a transformed host cell can be calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection Or by other methods. Such methods are described in many standard laboratory manuals such as, for example, Davis et al., Basic Methods In Molecular Biology (1986). Once a transformed host cell has been obtained, it has an assimilable source of carbon, nitrogen, and essential minerals that assists the growth of the host cell under any physiologically compatible conditions of pH and temperature. It can be cultured in any suitable nutrient medium, including. Recombinant polypeptide production culture conditions vary according to the type of vector used to transform the host cell. For example, a particular expression vector may require cell growth at a specific temperature or addition of a specific chemical or inducer to the cell growth medium to initiate gene expression that results in production of the recombinant polypeptide. Includes necessary regulatory regions. Thus, as used herein, the term “recombinant polypeptide production conditions” is not meant to be limited to any one set of culture conditions. Appropriate culture media and conditions for the above host cells and vectors are well known in the art. Following its production in the host cell, the polypeptide of interest can be isolated by several techniques. To release the polypeptide of interest from the host cell, the cell is lysed or destroyed. This lysis can be achieved by contacting the cells with a hypotonic solution, by treating with a cell wall disrupting enzyme such as lysozyme, by sonication, by treatment at high pressure, or a combination of the above methods. Can be achieved. Other bacterial cell disruption and lysis methods known to those skilled in the art can also be used.

  After disruption, the polypeptide can be separated from the cell debris by any technique suitable for separating particles in a complex mixture. The polypeptide can then be purified by well-known isolation techniques. Suitable techniques for purification include ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunosorption, anion exchange or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC), high performance LC (HPLC), fast performance LC (FPLC), hydroxyl apatite chromatography and lectin chromatography For example, but not limited to.

(kit)
The invention also provides kits for the synthesis, amplification, or sequencing of nucleic acid molecules. Kits according to this aspect of the invention can include one or more containers (eg, vials, tubes, ampoules, bottles, etc.), which can include one or more compositions of the invention.

  The kits of the invention may comprise one or more of the following components: (i) one or more compounds or compositions of the invention, (ii) one or more polymerases or reverse transcriptases, (iii) one or more suitable Buffer, (iv) one or more nucleotides, and (v) one or more primers.

  It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods and applications described herein will be apparent and can be made without departing from the scope of the invention or any embodiment thereof. It is easy to see. Although the present invention has been described in detail herein, the present invention will be more clearly understood by reference to the following examples, which are described herein for purposes of illustration only. Included and is not intended to limit the invention.

(Introduction)
4-Methylmorpholine N-oxide (hereinafter referred to as “MMNO”) was tested on a number of different PCR amplicons containing high GC content (eg, CAG repeats, Pseudomonas genomic DNA, etc.). The amplicons tested were difficult to amplify using a standard PCR reaction mixture that is accepted. To test the effectiveness of the new co-solvent in PCR runs from high GC content amplicons, MMNO and other co-solvents were added to the PCR reaction mixture at different concentrations.

(Method)
The following section describes the preparation of the chemicals used in the examples.

(Preparation of 2.2 × PCR mixture (Example 1-3))
In Example 1-3, a 2.2 × PCR mixture containing all the components listed below except for template DNA and primers was prepared. The following table exemplifies a method for preparing a 2.2 × PCR mixture.

（実施例１−９についての物質）：ベタイン一水和物（［カルボキシメチル］トリメチル−アンモニウム）、Ｌ−プロリン、４−メチルモルホリン−４−オキシド（ＭＭＮＯ）、エクトイン（ＴＨＰ［Ｂ］；［Ｓ］−２−メチル−１，４，５，６−テトラヒドロピリミジン−４−カルボン酸）、ＤＬ−ピペコリン酸（ＤＬ−２−ピペリジンカルボン酸）、およびＬ−カルニチン（［−］−ｂ−ヒドロキシ−ｇ−［トリメチルアンモニオ］酪酸塩）を、Ｓｉｇｍａ（Ｓｔ．Ｌｏｕｉｓ、ＭＯ）から購入し、そして滅菌蒸留水中に４Ｍストック溶液として調製し、そして濾過滅菌した。ＰＣＲ試薬：プラチナ（Ｐｌａｔｉｎｕｍ）Ｔａｑ ＤＮＡポリメラーゼ、プラチナＴａｑ ＤＮＡポリメラーゼ高忠実度、１０×Ｔａｑ緩衝液（１×＝２０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ８．４）、５０ｍＭ ＫＣｌ）、５０ｍＭ塩化マグネシウム、１０×Ｔａｑ高忠実度緩衝液（１×＝６０ｍＭ Ｔｒｉｓ−ＳＯ₄（ｐＨ８．９）、１８ｍＭ（ＮＨ₄）₂ＳＯ₄、５０ｍＭ硫酸マグネシウム、１０ｍＭ ｄＮＴＰ混合物、Ｋ５６２ヒトゲノムＤＮＡ，および滅菌蒸留水を、Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．（Ｒｏｃｋｖｉｌｌｅ、ＭＤ）から入手した。オリゴヌクレオチドプライマーを、Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．から脱塩調製物として購入し、そしてさらなる精製なしで使用した。

(Materials for Example 1-9): Betaine monohydrate ([carboxymethyl] trimethyl-ammonium), L-proline, 4-methylmorpholine-4-oxide (MMNO), ectoine (THP [B]; [ S] -2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid), DL-pipecolic acid (DL-2-piperidinecarboxylic acid), and L-carnitine ([-]-b-hydroxy -G- [trimethylammonio] butyrate) was purchased from Sigma (St. Louis, MO) and prepared as a 4M stock solution in sterile distilled water and filter sterilized. PCR reagents: Platinum Taq DNA polymerase, Platinum Taq DNA polymerase high fidelity, 10 × Taq buffer (1 × = 20 mM Tris-HCl (pH 8.4), 50 mM KCl), 50 mM magnesium chloride, 10 × Taq high Fidelity buffer (1 × = 60 mM Tris-SO ₄ (pH 8.9), 18 mM (NH ₄ ) ₂ SO ₄ , 50 mM magnesium sulfate, 10 mM dNTP mixture, K562 human genomic DNA, and sterile distilled water were added to Life Technologies, Inc. (Rockville, MD) Oligonucleotide primers were purchased as a desalted preparation from Life Technologies, Inc. and used without further purification.

Example 1: Titration of proline, 1-methylimidazole and 4-methylimidazole to improve PCR amplification from GC rich templates
Several chemicals were tested to see if they improved PCR performance using the GC rich template. In the first example, the GC rich template P55G12 was tested at various concentrations of one of the following three different chemicals: amino acids proline, 1-methylimidazole and 4-methylimidazole. The following components were combined in a 0.2 ml tube: 2.2 ml PCR mix 13 ml, template DNA 0.5 ml (10 pg), primer mix (10 mM each) 0.5 ml, and 4 M chemical solution (proline) , 1-methylimidazole or 4-methylimidazole) 13 ml. This solution was then mixed by pipetting. The PCR program was as follows: 95 ° C for 3 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 1 minute for 30 cycles. After performing PCR, 5 ml of loading dye was added to each tube and 12 ml of the mixture was loaded onto an electrophoresis agarose gel, which was then ethidium bromide stained for the presence of DNA fragments.

  As shown in FIG. 1, the specific concentration of each of these three chemicals performed better than the other concentrations. In the case of proline, 300-600 mM provided optimal amplification of P55G12, but concentrations higher than 600 mM did not provide any product. In the case of 1-methylimidazole, 100 mM and 200 mM worked best, but higher or lower concentrations either did not work at all or produced much less product. In the case of 4-methylimidazole, a slightly lower range of concentrations improved amplification: 60-100 mM. Note that in the absence of addition of these compounds to the PCR reaction, there was no amplification product and 1M betaine was effective in making the reaction productive.

Example 2: Titration of 4-methylmorpholine N-oxide (MMNO) for GC rich template PCR; comparison of MMNO and betaine
In Example 1, MMNO was identified as a novel reagent to improve PCR performance with GC rich templates. The next issue to be addressed was the dependence of MMNO performance on its concentration in the PCR mix. The GC rich amplicon P55G12 was amplified as described above in Example 1 in the presence of 1M betaine or various concentrations of MMNO.

  As shown in FIG. 2, the inclusion of 400-1000 mM MMNO in the PCR reaction mixture resulted in the production of a PCR product that was comparable in intensity to the production of the PCR product with 1M betaine. MMNO concentrations below 400 mM did not result in the generation of PCR products.

(Example 3: PCR amplification of high GC content amplicon: Pseudomonas aeruginosa amplicon)
The genome of Pseudomonas aeruginosa contains a high GC content (70% GC) and is therefore interesting for amplification by PCR. Thus, in the PCR method of the present invention using three different Pseudomonas aeruginosa amplicons ranging in size from 1.3 kb to 1.8 kb, using betaine, MMNO or trimethylamine N-oxide (TMANO) in the PCR reaction mixture. Tested. Template DNA in these reactions was 30 ng of P. aeruginosa. The same reaction preparation as described in Example 1 was used except that it consisted of aeruginosa genomic DNA. The following PCR program was used for DNA amplification: 35 cycles of 95 ° C. for 5 minutes; 94 ° C. for 2 minutes, 58 ° C. for 30 seconds, and 72 ° C. for 2 minutes.

  As shown in FIG. 3, sequence D of Pseudomonas aeruginosa was amplified in the presence of betaine or MMNO, while sequences E and F were amplified in the presence of betaine, MMNO or TMANO. These results indicate that long natural GC-rich sequences (eg, sequences from the genome of P. aeruginosa) can be efficiently amplified using the compositions and methods of the present invention.

Example 4: Comparison of betaine, proline, and MMNO for enhanced PCR amplification of GC rich templates
Varying concentrations of betaine, proline, and MMNO were added to the 156 bp fragment of human p53 exon 10 (62.2% GC) or the 2782 bp coding region for the DNA polymerase I gene from Deinococus radiodurans (66.7% GC). Were tested for their efficacy to enhance the PCR amplification. The effect of buffer composition and magnesium concentration was evaluated by varying the reaction parameters.

PCR amplification was performed using a thin walled 0.2 ml tube in a 50 ml reaction containing: 2.5 U platinum Taq, 1 × Taq buffer (20 mM Tris-HCl, pH 8.4), 50 mM KCl) or 1 × Taq high fidelity buffer (60 mM Tris-SO ₄ (pH 8.9), 18 mM (NH ₄ ) ₂ SO ₄ ), 200 mM each dNTP, and 200 nM each primer . As magnesium concentration, either magnesium chloride (Taq buffer reaction) or magnesium sulfate (Taq high fidelity buffer reaction) was varied between 1.0 mM and 2.5 mM. The amount of each co-solvent (betaine, MMNO, or proline) was varied as shown in each figure. The reaction was temperature cycled using either a Prekin Elmer model 9600 or 2400 thermal cycler. For amplification of human p53 exon 10 sequence, the reaction contains 100 ng of K562 human genomic DNA and denatured at 95 ° C. for 1 minute, then 95 ° C. for 30 seconds; 60 ° C. for 30 seconds annealing and 68 ° C. for 1 minute Incubated for 35 cycles of extension. For amplification of the DNA pol I gene, the reaction contains 20 ng of Deinococcus radiodurans genomic DNA and denatured at 95 ° C. for 1 minute, then 95 ° C. for 30 seconds; annealing at 55 ° C. for 30 seconds, and 3 at 68 ° C. Incubated with 35 cycles of minute extension. 10 ml of each PCR was analyzed for the presence of the expected DNA fragment by agarose gel electrophoresis and ethidium bromide staining.

As shown in FIG. 4, the successful amplification of the 156 bp human p53 sequence was dependent on the magnesium concentration and the specific buffer conditions. In the absence of added cosolvent, no specific product was detected in the reaction containing standard PCR buffer and ammonium sulfate buffer (Taq high fidelity buffer only with 1.0 mM MgSO _4. ) Was detectable in PCR. Addition of betaine, MMNO or proline cosolvent improved the specificity and yield of the amplification product over a wider range of magnesium concentrations in both buffer systems. The effect of MMNO in the optimum magnesium concentration range was less evident than the effect of either betaine or proline. The concentration of betaine or proline producing a broad magnesium optimum was higher in Taq buffer than Taq high fidelity buffer.

  In contrast to the results obtained with amplification of p53 exon 10, MMNO enhances the longer 2.8 kb amplicon PCR for Dra DNA pol I over a broad magnesium concentration range of 1.0-2.5 mM. It was highly effective to do this (FIG. 5). This effect was obtained for MMNO concentrations between 0.4M and 0.8M and was similar to the effect observed for 1M betaine. Addition of proline was also effective in enhancing amplification of the DNA pol I fragment: however, the effective concentration range for proline is much narrower and its effect on the magnesium concentration range is observed for betaine or MMNO The effect was not as obvious. In general, higher concentrations of each co-solvent were required to enhance PCR in standard Taq buffer reactions than in reactions containing Taq high fidelity buffer. This is consistent with the results obtained for the amplification of p53 exon 10. No Dra DNA pol I PCR product was observed in the reaction without co-solvent.

(Example 5: MMNO and proline mixture enhances PCR amplification of GC rich template)
The results of the previous examples show that proline is highly effective in enhancing the optimal conditions of reaction for amplification of short GC-rich templates, and that MMNO is effective in enhancing amplification of long GC-rich templates. This mixture of the two compounds was tested to see if they provide enhanced GC-rich template amplification regardless of fragment size. To test this possibility, the GC rich template was amplified in the presence of a composition containing proline, MMNO, or both.

  In the mixture composition, 4M solutions of MMNO and proline were combined in ratios of 3: 1, 2: 1, 1: 1, 1: 2, and 1: 3, respectively, to form a 4M hybrid co-solvent mixture. These mixtures were then assayed for effects on PCR amplification of p53 exon 10 and Dra DNA pol I. PCR reactions were performed in 50 ml volumes with platinum Taq DNA polymerase in 1 × Taq high fidelity buffer as described above. Magnesium sulfate concentration was varied between 1.0 and 2.5 mM for each concentration of co-solvent tested. The concentrations of MMNO, a mixture of MMNO and proline, proline, and betaine are as shown in each figure.

  As shown in FIG. 6, the mixture of MMNO and proline enhances the specific amplification of the 156 bp p53 exon 10 fragment over the wider magnesium concentration range and cosolvent concentration range obtained with either cosolvent alone. It was effective. The use of the MMNO: proline mixture is also highly effective in facilitating amplification of the 2.8 kb Dra DNA pol I fragment and reduces the effective magnesium concentration range and cosolvent concentration range over those obtained with proline alone. Significantly increased (FIG. 7). As previously observed, MMNO enhanced PCR amplification over the entire range of magnesium and cosolvent concentrations tested. Collectively, these results give rise to novel properties that the use of a composition comprising a mixture of N-alkyl carboxylic acids and N-alkylamine oxides can be exploited to enhance PCR amplification of GC-rich templates. Prove that. In particular, a mixture of MMNO and proline combined in a ratio of 2: 1 to 1: 2 can be used to enhance PCR amplification of GC-rich templates over a wide range of sizes, and a wider range of magnesium The reliability of PCR amplification can be increased over concentration.

Example 6: MMNO: proline mixture enhances GC-rich template PCR over a wide range of annealing temperature optimums
To evaluate the effect of the PCR co-solvent on the optimal annealing temperature during the PCR reaction, the GC rich template P32D9 was amplified by PCR using the MMNO: proline mixture composition described above. PCR reactions were performed in 50 ml volumes using thin walled 0.2 ml tubes (Stratagene, Inc.) in a 50 ml reaction containing: 2.5 U platinum Taq, 1 × Taq high fidelity buffer. (60 mM Tris-SO ₄ (pH 8.9), 18 mM (NH ₄ ) ₂ SO ₄ ), 1.5 mM MgSO ₄ , 200 mM each dNTP, 200 nM each primer, 100 ng K562 human genomic DNA, and 0. Either 5M betaine or 0.5M 1: 1 MMNO: proline added co-solvent or no added co-solvent. A concentrated MMNO: proline mixture was prepared by mixing equal volumes of 4M MMNO and 4M proline. The optimum conditions of annealing temperature were studied using a gradient block Robo-cycler (Stratagene) with a heated lid for oil-free operation. Following denaturation at 95 ° C for 1 minute, the reaction was cycled 35 times at 95 ° C for 45 seconds; 55-66 ° C for 45 seconds, 68 ° C for 1 minute. Ten ml of each PCR was analyzed by agarose gel electrophoresis (1% agarose 1000, Life Technologies, Inc.) in 0.5 × TBE, and ethidium bromide staining.

  As shown in FIG. 8, in the absence of PCR co-solvent, a specific PCR product (149 bp, 78.5% GC fragment) was obtained only at 66 ° C. As the annealing temperature was decreased, the product yield decreased rapidly, resulting in amplification of non-specific products. In contrast, both betaine and MMNO: proline mixtures extended the range of effective annealing temperatures. The use of the MMNO: proline mixture resulted in a product yield higher than that obtained with betaine and allowed the detection of specific products over all annealing temperature gradients from 66 ° C to 55 ° C.

Example 7: Use of MMNO: proline mixture for amplification of fragile X CGG repeats
The MMNO: proline mixture was compared to betaine for its ability to promote PCR amplification of DNA sequences with very high GC content. The primer was designed to contain a CGG repeat of the human FMR-1 gene (GenBank accession number X61378) at the fragile X locus. PCR amplification was performed as described above using 1 × Taq high fidelity buffer supplemented with 2.5 U platinum Taq DNA polymerase high fidelity, 100 ng K562 genomic DNA, and 2 mM magnesium sulfate (final concentration). Carried out. An aliquot of 4M MMNO: proline mixture, prepared as above, or 4M betaine was added to the PCR in varying amounts to produce a final concentration of 0.25M-2M of either co-solvent. . The PCR was incubated at 35 ° C. for 1 minute, then at 95 ° C. for 30 seconds; 58 ° C. for 30 seconds; 68 ° C. for 30 seconds. The agarose gel analysis of these study results is shown in FIG.

  The results of these studies demonstrate the superior ability of the MMNO: proline mixture to promote PCR amplification of target sequences with extremely high (> 80%) GC content when compared to betaine. In the absence of PCR co-solvent, no specific PCR product was detected. However, in the reaction containing 1.75 M betaine, a small band of the expected size could be recognized. In contrast, robust amplification of CGG repeats was demonstrated in reactions containing 1.5-2 M MMNO: proline.

Example 8: Use of MMNO: proline mixture in long PCR
A DNA polymerase mixture consisting of Taq DNA polymerase and archaeal DNA polymerase possessing proofreading activity has been used for amplification of DNA fragments up to 40 kb (Barnes, WM Proc. Natl. Acad. Sci. USA 91: 2216-2220 (1994)). The ability of the MMNO: proline mixture to promote the amplification of long GC-rich sequences can be achieved by using primers designed to amplify a 7.77 kb or 9.75 kb fragment (approximately 60% GC) of type 2 adenovirus DNA. Used and tested. PCR is essential except that instead of platinum Taq DNA polymerase, 2.5U platinum Taq DNA polymerase high fidelity (which is an enzyme mixture of DNA polymerase from Thermus aquaticus and Pyrococcus sp. GB-D) As described above, 1 pg of type 2 adenovirus DNA (Life Technologies, Inc.), 1 × Taq high fidelity buffer supplemented with 1.5 mM magnesium sulfate, and varying amounts (0-1 M) of MMNO. : Performed using proline mixture. The reaction was incubated at 35 ° C. for 1 minute, followed by 35 cycles of 95 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 68 ° C. for 10 minutes.

  As shown in FIG. 10, the successful amplification of the expected 7.77 kb and 9.75 kb DNA fragments was dependent on the inclusion of the MMNO: proline cosolvent. No specific product was detected in the reaction without MMNO: proline; however, containing 0.25 M MMNO: proline mixture resulted in robust amplification and high product yield . The product yield in long PCR is sensitive to the amount of MMNO: proline used so that the product yield for both 7.3 kb and 9.7 kb targets decreases as the concentration of MMNO: proline increases. there were.

Example 9: Comparison of compensation solutes to enhance PCR amplification of GC rich templates
A wide variety of amino compounds have been shown to perform important functions in protecting organisms from osmotic stress. Betaine and proline are E. coli. The major osmoprotectant in E. coli. Since both of these compounds disrupt the stability of the DNA helix, thereby facilitating the amplification of the GC rich template, the effect of other known and commercially available osmotic compounds on PCR was studied.

The PCR mixture was prepared in a 50 ml volume containing: 2.5 U platinum Taq DNA polymerase, 60 mM Tris-SO ₄ (pH 8.9), 18 mM (NH ₄ ) ₂ SO ₄ , 1.5 mM MgSO. ₄ , 200 mM dNTP (each), 200 nM primer (each), 100 ng K562 human genomic DNA. A varying amount of PCR co-solvent was then prepared. The reaction was incubated for 35 cycles of 95 ° C for 1 minute, then 95 ° C for 30 seconds; 58 ° C for 30 seconds; 68 ° C for 1 minute.

  A comparison of the efficacy of the MMNO: proline mixture, betaine, L-carnitine, and DL-pipecolic acid is shown in FIG. On the other hand, the result of a separate experiment comparing ectoine to betaine is shown in FIG. The experimental conditions were essentially the same as described above, except that the final reaction volume was 25 ml.

  All osmolytes tested in these experiments are effective to allow amplification of the P32D9 sequence, and a wide variety of N-alkyl carboxylic acid derivatives are difficult templates (eg, It demonstrates that it can be used to promote PCR amplification of templates with high GC content.

  Although the present invention has been described fully in some detail by way of illustration and example for purposes of clarity of understanding, the same will not affect the scope of the invention or any particular embodiment thereof. Can be practiced by modifying or altering the present invention within the broad and equivalent scope of conditions, formulations, and other parameters, and such modifications or changes are encompassed within the scope of the appended claims. It will be apparent to those skilled in the art that

  All publications, patents, and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and each individual publication, patent, or patent application is unambiguous. And to the same extent as if individually indicated to be incorporated by reference.

Claims (29)

A composition for use in the synthesis of a nucleic acid molecule, wherein the composition has one or more compounds having a chemical formula selected from the group consisting of the following formula I or formula II, or a salt or A composition comprising a derivative comprising:

Where A is

Is;
Where X is

Is;
Where q = 1 to 100,000, where q = 2 to 100,000, each monomer of formula I may be the same as or different from other monomers of formula I ;
Where Z may be the same as or different from Y;
Wherein each Y and Z is independently selected from the group consisting of —OH, —NH ₂ , —SH, —PO ₃ H, —CO ₂ H, —SO ₃ H and hydrogen;
Where f is an integer from 0 to 2, m is an integer from 0 to 20, and e is an integer from 0 to 2;
Where R ₄ , R ₅ and R ₆ may be the same or different and are hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, mercaptan, thiol, halo, nitro, nitrilo, hydroxy, hydroxyalkyl Independently selected from the group consisting of: hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, carbonyl, sulfonyl, sulfone and amide groups, and d is an integer from 0 to 2;
Where a, b and c are independently integers from 0 to 1, provided that more than two of a, d and c are not 0;
Here, R ₁ , R ₂ and R ₃ may be the same or different and are:
a) = O;
b)

Independently selected from the group consisting of:
Where each R ₇ and W may be the same or different and are hydrogen, alkyl, alkenyl, alkynyl, aryl, amino, thiol, mercaptan, halo, nitro, nitrilo, hydroxy, hydroxyalkyl, hydroxy Independently selected from the group consisting of aryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, carbonyl, sulfonyl, sulfone and amide groups; g is an integer from 0 to 2 and n is 0 An integer of ~ 20;

Where formula II is saturated or unsaturated;
Where q = 1-100,000, where q = 2-100,000, each monomer of formula II may be the same as or different from the other monomers of formula II;
Where X is selected from the group consisting of N, C, O, P and S;
Here, Y is O, N, S, P, C, —O—NH—, —O—CH ₂ —NH—, —O—CH ₂ —O—, —NH—CH ₂ —NH—, —O. —CH (CH ₃ ) —NH—, —NH—CH (CH ₃ ) —NH—, —O—CH (CH ₃ ) —O—, —NH—C (CH ₃ ) ₂ —NH—, —O— _{S -, - O-CH 2} -S -, - NH-S -, - NH-CH 2 -S- and other mercaptans, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, sulfonyl, sulfone And selected from the group consisting of amide groups;
Where R ₁ , R ₂ , R ₃ , R ₄ , R ₅ , R ₆ , R ₇ , and R ₈ may be the same or different and are hydrogen, alkyl, alkenyl, alkynyl, aryl Independently of the group consisting of, amino, thiol, mercaptan, halo, nitro, nitrilo, hydroxy, hydroxyalkyl, hydroxyaryl, phosphato, alkoxy, oxide, ether, ester (alkanoyloxy), carboxy, sulfonyl, sulfone and amide groups And where a, b, c, d, e, m, n, and o are integers that may be the same or different, and a, b, c, d, and e Independently selected from 0 to 2 and m, n and o are independently selected from 0 to 5;
Composition. A composition according to claim 1, provided that, q = 1 and _{(R 1) a, (R} 2) b, and (R ₃₎ one of _c but is oxygen and the other two are the same A composition wherein A is not methyl, ethyl or propyl, if or different and selected independently from the group consisting of hydrogen, methyl, ethyl and propyl. 2. The composition of claim 1, wherein when a, b, or c is 0, the corresponding R group is an electron pair. The composition of claim 1, wherein Y and / or X is N and m, n and o are 1. The composition of claim 1, wherein Y and / or X is N and / or O, m and n are 1, and o is 2. 2. The composition of claim 1, wherein the composition comprises at least two compounds having formula I or II, or salts or derivatives thereof. Said composition comprises 2 to 5 compounds having formula I or II, or salts or derivatives thereof. The composition according to claim 6. The composition of claim 6, wherein the composition comprises proline or a derivative thereof. The composition according to claim 6, wherein the composition comprises an N-alkylimidazole compound. The composition according to claim 9, wherein the N-alkylimidazole compound is 1-methylimidazole or 4-methylimidazole. The compound includes 4-methylmorpholine N-oxide, betaine, carnitine, ectoine, poly (2-ethyl-2-oxazoline) having an average molecular weight of about 50,000 to about 500,000 daltons, and an average molecular weight of about 100. The composition of claim 1 selected from the group consisting of from 1,000,000 to about 200,000 daltons of poly (diallyldimethylammonium chloride). The compound includes 4-methylmorpholine N-oxide, betaine, carnitine, ectoine, poly (2-ethyl-2-oxazoline) having an average molecular weight of about 50,000 to about 500,000 daltons, and an average molecular weight of about 100,000 to 7. The composition of claim 6, wherein the composition is selected from the group consisting of about 200,000 daltons poly (diallyldimethylammonium chloride). 2. The composition of claim 1, further comprising one or more enzymes having nucleic acid polymerase activity. 7. The composition of claim 6, further comprising one or more enzymes having nucleic acid polymerase activity. 14. The composition of claim 13, wherein the enzyme having nucleic acid polymerase activity is selected from the group consisting of DNA polymerase, RNA polymerase and reverse transcriptase. 15. The composition of claim 14, wherein the enzyme having nucleic acid polymerase activity is selected from the group consisting of DNA polymerase, RNA polymerase and reverse transcriptase. 16. The composition of claim 15, wherein the DNA polymerase is selected from the group consisting of Taq, Tne, Tma, Pfu, VENT ^™ , DEEPVENT ^™ , and Tth DNA polymerase, and variants, variants and derivatives thereof. The reverse transcriptase is from M-MLV reverse transcriptase, RSV reverse transcriptase, AMV reverse transcriptase, RAV reverse transcriptase, MAV reverse transcriptase, and HIV reverse transcriptase, and variants, variants and derivatives thereof. The composition of claim 15, wherein the composition is selected from the group consisting of: 16. The composition of claim 15, wherein the reverse transcriptase has a substantially reduced RNase H activity. Use in synthesizing nucleic acid molecules comprising one or more components selected from the group consisting of one or more amino acids, one or more saccharides, one or more polyalcohols, or derivatives thereof, or combinations thereof For the composition. 21. A composition obtained by combining two or more compounds or components according to any one of claims 1 or 20. A method for synthesizing a nucleic acid molecule, the method comprising:
(A) mixing a nucleic acid template with one or more compositions of claim 1 or 20 to form a mixture; and (b) a first complementary to all or part of the template. Incubating the mixture under conditions sufficient to produce a nucleic acid molecule;
Including the method. 23. The method of claim 22, further comprising incubating the first nucleic acid molecule under conditions sufficient to produce a second nucleic acid molecule that is complementary to all or a portion of the first nucleic acid molecule. the method of. 23. A nucleic acid molecule made according to the method of claim 22. A method for amplifying a nucleic acid molecule comprising the following:
(A) mixing a nucleic acid template with one or more compositions of claim 1 or 20 to form a mixture; and (b) a nucleic acid molecule complementary to all or part of the template. Incubating the mixture under conditions sufficient to amplify;
Including the method. A method for sequencing a nucleic acid molecule comprising the following:
(A) mixing the nucleic acid molecule to be sequenced with one or more primers, one or more compositions according to claim 1 or 20, one or more nucleotides, and one or more terminators; Forming a mixture;
(B) incubating the mixture under conditions sufficient to synthesize a population of molecules complementary to all or part of the molecule to be sequenced; and (c) separating the population; Determining the nucleotide sequence of all or part of the molecule to be sequenced;
Including the method. 21. A kit for use in synthesizing nucleic acid molecules, wherein the kit comprises one or more compounds or components of claim 1 or 20. 28. The kit of claim 27, wherein the kit comprises at least two of the compounds or components. The kit comprises the group consisting of one or more nucleotides, one or more DNA polymerases, one or more transcriptases, one or more suitable buffers, one or more primers, and one or more terminators. 28. The kit of claim 27, further comprising one or more selected components.

Images